Edg receptor agonists

ABSTRACT

The present invention encompasses compounds of Formula 1: as well as the pharmaceutically acceptable salts and hydrates thereof. The compounds are useful for treating immune mediated diseases and conditions, such as bone marrow, organ and tissue transplant rejection. Pharmaceutical compositions and methods of use are included.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase application under 35 U.S.C. §371 of PCT Application No. US03/01196, filed Jan. 15, 2003, which claimspriority under 35 U.S.C. 119 to U.S. Ser. No. 60/350,000, filed Jan. 18,2002.

BACKGROUND OF THE INVENTION

The present invention is related to compounds that are S1P₁/Edg1receptor agonists and thus have immunosuppressive activities byproducing lymphocyte sequestration in secondary lymphoid tissues. Theinvention is also directed to pharmaceutical compositions containingsuch compounds and methods of treatment or prevention.

Immunosuppressive agents have been shown to be useful in a wide varietyof autoimmune and chronic inflammatory diseases, including systeniclupus erythematosis, chronic rheumatoid arthritis, type I diabetesmellitus, inflammatory bowel disease, biliary cirrhosis, uveitis,multiple sclerosis and other disorders such as Crohn's disease,ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis,autoimmune myositis, Wegener's granulomatosis, ichthyosis, Gravesophthalmopathy, atopic dermatitis and asthma. They have also proveduseful as part of chemotherapeutic regimens for the treatment ofcancers, lymphomas and leukemias.

Although the underlying pathogenesis of each of these conditions may bequite different, they have in common the appearance of a variety ofautoantibodies and/or self-reactive lymphocytes. Such self-reactivitymay be due, in part, to a loss of the homeostatic controls under whichthe normal immune system operates. Similarly, following a bone-marrow oran organ transplantation, the host lymphocytes recognize the foreigntissue antigens and begin to produce both cellular and humoral responsesincluding antibodies, cytokines and cytotoxic lymphocytes which lead tograft rejection.

One end result of an autoimmune or a rejection process is tissuedestruction caused by inflammatory cells and the mediators they release.Anti-inflammatory agents such as NSAIDs act principally by blocking theeffect or secretion of these mediators but do nothing to modify theimmunologic basis of the disease. On the other hand, cytotoxic agents,such as cyclophospharide, act in such a nonspecific fashion that boththe normal and autoimmune responses are shut off. Indeed, patientstreated with such nonspecific immunosuppressive agents are as likely tosuccumb to infection as they are to their autoimmune disease.

Cyclosporin A is a drug used to prevent rejection of transplantedorgans. FK-506 is another drug approved for the prevention of transplantorgan rejection, and in particular, liver transplantation. Cyclosporin Aand FK-506 act by inhibiting the body's immune system from mobilizingits vast arsenal of natural protecting agents to reject the transplant'sforeign protein. Cyclosporin A was approved for the treatment of severepsoriasis and has been approved by European regulatory agencies for thetreatment of atopic dermatitis.

Though they are effective in delaying or suppressing transplantrejection, Cyclosporin A and FK-506 are known to cause severalundesirable side effects including nephrotoxicity, neurotoxicity, andgastrointestinal discomfort. Therefore, an immunosuppressant withoutthese side effects still remains to be developed and would be highlydesirable.

The immunosuppressive compound FTY720 is a lymphocyte sequestrationagent currently in clinical trials. FTY720 is metabolized in mammals toa compound that is a potent agonist of sphingosine 1-phosphatereceptors. Agonism of sphingosine 1-phosphate receptors induces thesequestration of lymphocytes (T-cells and B-cells) in lymph nodes andPeyer's patches without lymphodepletion. Such immunosuppression isdesirable to prevent rejection after organ transplantation and in thetreatment of autoimmune disorders.

Sphingosine 1-phosphate is a bioactive sphingolipid metabolite that issecreted by hematopoietic cells and stored and released from activatedplatelets. Yatomi, Y., T. Ohmori, G. Rile, F. Kazama, H. Okamoto, T.Sano, K. Satoh, S. Kume, G. Tigyi, Y. Igarashi, and Y. Ozaki. 2000.Blood. 96:3431-8. It acts as an agonist on a family of G protein-coupledreceptors to regulate cell proliferation, differentiation, survival, andmotility. Fukushima, N., I. Ishii, J. J. A. Contos, J. A. Weiner, and J.Chun. 2001. Lysophospholipid receptors. Annu. Rev. Pharmacol. Toxicol.41:507-34; Hla, T., M.-J. Lee, N. Ancellin, J. H. Paik, and M. J. Kluk.2001. Lysophospholipids—Receptor revelations. Science. 294:1875-1878;Spiegel, S., and S. Milstien. 2000. Functions of a new family ofsphingosine-1-phosphate receptors. Biochim. Biophys. Acta. 1484:107-16;Pyne, S., and N. Pyne. 2000. Sphingosine 1-phosphate signalling via theendothelial differentiation gene family of G-protein coupled receptors.Pharm. & Therapeutics. 88:115-131. Five sphingosine 1-phosphatereceptors have been identified (S1P₁, S1P₂, S1P₃, S1P₄, and S1P₅, alsoknown as endothelial differentiation genes Edg1, Edg5, Edg3, Edg6,Edg8), that have widespread cellular and tissue distribution and arewell conserved in human and rodent species (see Table). Binding to S1Preceptors elicits signal transduction through Gq-, Gi/o, G12-, G13-, andRho-dependent pathways. Ligand-induced activation of S1P₁ and S1P₃ hasbeen shown to promote angiogenesis, chemotaxis, and adherens junctionassembly through Rac- and Rho-, see Lee, M.-J., S. Thangada, K. P.Claffey, N. Ancellin, C. H. Liu, M. Kluk, M. Volpi, R. I. Sha'afi, andT. Hla. 1999. Cell. 99:301-12, whereas agonism of S1P₂ promotes neuriteretraction, see Van Brocklyn, J. R., Z. Tu, L. C. Edsall, R. R. Schmidt,and S. Spiegel. 1999. J. Biol. Chem. 274:4626-4632, and inhibitschemotaxis by blocking Rac activation, see Okamoto, H., N. Takuwa, T.Yokomizo, N. Sugimoto, S. Sakurada, H. Shigematsu, and Y. Takuwa. 2000.Mol. Cell. Biol. 20:9247-9261. S1P₄ is localized to hematopoietic cellsand tissues, see Graeler, M. H., G. Bernhardt, and M. Lipp. 1999. Curr.Top. Microbiol. Immunol. 246:131-6, whereas S1P₅ is primarily a neuronalreceptor with some expression in lymphoid tissue, see Im, D. S., C. E.Heise, N. Ancellin, B. F. O'Dowd, G. J. Shei, R. P. Heavens, M. R.Rigby, T. Hla, S. Mandala, G. McAllister, S. R. George, and K. R. Lynch.2000. J. Biol. Chem. 275:14281-6. Administration of sphingosine1-phosphate to animals induces systemic sequestration of peripheralblood lymphocytes into secondary lymphoid organs, stimulatesFGF-mediated blood vessel growth and differentiation, see Lee, et al.,supra, but also has cardiovascular effects that limit the utility ofsphingosine 1-phosphate as a therapeutic agent, see Sugiyama, A., N. N.Aye, Y. Yatomi, Y. Ozaki, and K. Hashimoto. 2000. Jpn. J. Pharmacol.82:338-342. The reduced heart rate and blood pressure measured withsphingosine 1-phosphate is associated with its non-selective, potentagonist activity on all S1P receptors.

The present invention encompasses compounds which are agonists of theS1P₁/Edg1 receptor having selectivity over the S1P₃/Edg3 receptor. AnS1P₁/Edg1 receptor selective agonist has advantages over currenttherapies and extends the therapeutic window of lymphocytessequestration agents, allowing better tolerability with higher dosingand thus improving efficacy as monotherapy.

While the main use for immunosuppressants is in treating bone marrow,organ and transplant rejection, other uses for such compounds includethe treatment of arthritis, in particular, rheumatoid arthritis, insulinand non-insulin dependent diabetes, multiple sclerosis, psoriasis,inflammatory bowel disease, Crohn's disease, lupus erythematosis and thelike.

Thus, the present invention is focused on providing immunosuppressantcompounds that are safer and more effective than prior compounds. Theseand other objects will be apparent to those of ordinary skill in the artfrom the description contained herein.

Summary of S1P receptors Coupled G Name Synonyms proteins mRNAexpression S1P₁ Edg1, LP_(B1) G_(i/o) Widely distributed, endothelialcells S1P₂ Edg5, LP_(B2), G_(i/o), G_(q), Widely distributed, vascularAGR16, H218 G_(12/13) smooth muscle cells S1P₃ Edg3, LP_(B3) G_(i/o),G_(q), Widely distributed, G_(12/13) endothelial cells S1P₄ Edg6,LP_(C1) G_(i/o) Lymphoid tissues, lymphocytic cell lines S1P₅ Edg8,LP_(B4), NRG1 G_(i/o) Brain, spleen

SUMMARY OF THE INVENTION

The present invention encompasses compounds of Formula I:

as well as the pharmaceutically acceptable salts and hydrates thereof.The compounds are useful for treating immune mediated diseases andconditions, such as bone marrow, organ and tissue transplant rejection.Pharmaceutical compositions and methods of use are included.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses compounds represented by Formula I:

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

-   Ar is phenyl or naphthyl;-   m=0 or 1;-   n=0 or 1;-   A is selected from the group consisting of: —CO₂H, —PO₃H₂, —PO₂H,    —SO₃H, —PO(C₁₋₃alkyl)OH and 1H-tetrazol-5-yl;-   R¹ and R² are each independently selected from the group consisting    of: hydrogen, halo, hydroxy, —CO₂H and C₁₋₄alkyl, optionally    substituted from one up to the maximum number of substitutable    positions with halo;-   R³ is selected from the group consisting of: hydrogen and C₁₋₄alkyl,    optionally substituted with from one up to the maximum number of    substitutable positions with a substituent independently selected    from the group consisting of: halo and hydroxy;-   each R⁴ is independently selected from the group consisting of:    halo, C₁₋₄alkyl and C₁₋₃alkoxy, said C₁₋₄alkyl and C₁₋₃alkoxy    optionally substituted from one up to the maximum number of    substitutable positions with halo,-   C is selected from the group consisting of:    -   (1) C₁₋₈alkyl, C₁₋₈alkoxy, —(C═O)—C₁₋₆alkyl or —CHOH—C₁₋₆alkyl,        said C₁₋₈alkyl, C₁₋₈alkoxy, —(C═O)—C₁₋₆alkyl and —CHOH—C₁₋₆alkyl        optionally substituted with phenyl, and    -   (2) phenyl or HET, each optionally substituted with 1-3        substituents independently selected from the group consisting        of: halo, phenyl, C₁₋₄alkyl and C₁₋₄alkoxy, said C₁₋₄alkyl and        C₁₋₄alkoxy groups optionally substituted from one up to the        maximum number of substitutable positions with a substituent        independently selected from halo and hydroxy, and said phenyl        optionally substituted with 1 to 5 groups independently selected        from the group consisting of: halo and C₁₋₄alkyl, optionally        substituted with 1-3 halo groups,-   or C is not present;    when C is not present then B is selected from the group consisting    of: phenyl, C₅₋₁₆alkyl, C₅₋₁₆alkenyl, C₅₋₁₆alkynyl,    —CHOH—C₄₋₁₅alkyl, —CHOH—C₄₋₁₅alkenyl, —CHOH—C₄₋₁₅alkynyl,    C₄₋₁₅alkoxy, —O—C₄₋₁₅alkenyl, —O—C₄₋₁₅alkynyl, C₄₋₁₅alkylthio,    —S—C₄₋₁₅alkenyl, —S—C₄₋₁₅alkynyl, —CH₂—C₃₋₁₄alkoxy,    —CH₂—O—C₃₋₁₄alkenyl, —CH₂—O—C₃₋₁₄alkynyl, —(C═O)—C₄₋₁₅alkyl,    —(C═O)—C₄₋₁₅alkenyl, —(C═O)—C₄₋₁₅alkynyl, —(C═O)—O—C₃₋₁₄alkyl,    —(C═O)—O—C₃₋₁₄alkenyl, —(C═O)—O—C₃₋₁₄alkynyl,    —(C═O)—N(R⁶)(R⁷)—C₃₋₁₄alkyl, —(C═O)—N(R⁶)(R⁷)—C₃₋₁₄alkenyl,    —(C═O)—N(R⁶)(R⁷)—C₃₋₁₄alkynyl, —N(R⁶)(R⁷)—(C═O)—C₃₋₁₄alkyl,    —N(R⁶)(R⁷)—(C═O)—C₃₋₁₄alkenyl and —N(R⁶)(R⁷)—(C═O)—C₃₋₁₄alkynyl,-   when C is phenyl or HET then B is selected from the group consisting    of: C₁₋₆alkyl, C₁₋₅alkoxy, —(C═O)—C₁₋₅alkyl, —(C═O)—O—C₁₋₄alkyl,    —(C═O)—N(R⁶)(R⁷)—C₁₋₄alkyl,

-   when C is C₁₋₈alkyl, C₁₋₈alkoxy, —(C═O)—C₁₋₆alkyl or —CHOH—C₁₋₆alkyl    then B is phenyl; and-   R⁶ and R⁷ are independently selected from the group consisting of:    hydrogen, C₁₋₉alkyl and —(CH₂)_(p)-phenyl, wherein p is 1 to 5 and    phenyl is optionally substituted with 1-3 substituents independently    selected from the group consisting of: C₁₋₃alkyl and C₁₋₃alkoxy,    each optionally substituted with 1-3 halo groups.

An embodiment of the invention encompasses a compound of Formula Iwherein:

-   Ar is phenyl;-   the group —B—C is attached to the phenyl ring at the 3- or    4-position;-   C is phenyl or HET, each optionally substituted with 1-3    substituents independently selected from the group consisting of:    halo, phenyl, C₁₋₄alkyl and C₁₋₄alkoxy, said C₁₋₄alkyl and    C₁₋₄alkoxy groups optionally substituted from one up to the maximum    number of substitutable positions with a substituent independently    selected from halo and hydroxy, and said phenyl optionally    substituted with 1 to 5 groups independently selected from the group    consisting of: halo and C₁₋₄alkyl, optionally substituted with 1-3    halo groups,-   or C is not present;-   when C is not present then B is selected from the group consisting    of: C₇₋₁₂alkyl, C₇₋₁₂alkenyl, C₇₋₁₂alkynyl, C₆₋₁₁alkoxy,    —O—C₆₋₁₁alkenyl, —O—C₆₋₁₁alkenyl, —(C═O)—C₆₋₁₁alkyl,    —(C═O)—C₆₋₁₁alkenyl, —(C═O)—C₆₋₁₁alkynyl, —(C═O)—O—C₅₋₁₀alkyl,    (C═O)—O—C₅₋₁₉alkenyl, and —(C═O)—O—C₅₋₁₀alkynyl and C is not    present; and-   when C is phenyl or HET then B is selected from the group consisting    of C₁₋₅alkyl, C₁₋₄alkoxy, —(C═O)—C₁₋₄alkyl, —(C═O)—O—C₁₋₃alkyl,    phenyl and HET.

For purposes of this specification, when the group —B—C is attached tothe phenyl ring at the 3- or 4-position, it means the positions shown inthe following:

For purposes of this specification, C may be substituted at anysubstitutable position on B. For example, when B is methoxy and C isthiophene, thiophene replaces a hydrogen on the methoxy group. Furthervariations are illustrated in the examples that follow. Also, the pointof any attachments shown for B is to the Ar group. For example, when Bis —(C═O)—C₆₋₁₁alkynyl this means B is attached to Ar as follows:Ar—(C═O)—C₆₋₁₁alkynyl. C may then be substituted at any substituableposition on B.

An embodiment of the invention encompasses the compound of Formula Iwherein HET is selected from the group consisting of:

Another embodiment encompasses the compound of Formula I wherein m is 0.

Another embodiment encompasses the compound of Formula I wherein m is 1.

Another embodiment encompasses the compound of Formula I wherein n is 0.

Another embodiment encompasses the compound of Formula I wherein n is 1.

Another embodiment encompasses the compound of Formula I wherein B isselected from the group consisting of: C₇₋₁₂alkyl, C₇₋₁₂alkenyl,C₇₋₁₂alkynyl, C₆₋₁₁alkoxy, —O—C₆₋₁₁alkenyl, —O—C₆₋₁₁alkynyl,—(C═O)—C₆₋₁₁alkyl, —(C═O)—C₆₋₁₁alkenyl, —(C═O)—C₆₋₁₁alkynyl,—(C═O)—O—C₅₋₁₀alkyl, —(C═O)—O—C₅₋₁₉alkenyl, and —(C═O)—O—C₅₋₁₀alkynyland C is not present;

Another embodiment of the invention encompasses the compound of FormulaI wherein: B is methoxy and C is HET substituted with phenyl andC₁₋₄alkyl, said C₁₋₄alkyl optionally substituted from one up to themaximum number of substitutable positions with halo, and said phenyl,optionally substituted with 1 to 5 substituents independently selectedfrom the group consisting of: halo and C₁₋₄alkyl, optionally substitutedwith 1-3 halo groups. Within this embodiment is encompassed the compoundof Formula I wherein C is selected from the group consisting of:

Also encompassed is a compound of Formula I wherein C is thiophene orfuran.

Another embodiment of the invention encompasses the compound of FormulaI wherein: B is methoxy and C is HET. Within this embodiment isencompassed the compound of Formula I wherein C is selected from thegroup consisting of:

Also within this embodiment is encompassed the compound of Formula Iwherein C is benzothiophene or benzofuran.

Another embodiment of the invention encompasses the compound of FormulaI wherein: B is methoxy and C is phenyl substituted with two C₁₋₄alkylgroups, said C₁₋₄alkyl optionally substituted from one up to the maximumnumber of substitutable positions with halo.

Another embodiment of the invention encompasses the compound accordingto claim 1 wherein: B is HET and C is HET substituted with phenyl andC₁₋₄alkyl, said C₁₋₄alkyl optionally substituted from one up to themaximum number of substitutable positions with halo, and said phenyloptionally substituted with 1 to 5 substituents independently selectedfrom the group consisting of: halo, C₁₋₄alkyl, optionally substitutedwith 1-3 halo groups. Within this embodiment is encompassed the compoundof Formula I wherein B is 1,2,4-oxadiazole. Also within this embodimentis encompassed the compound of Formula I wherein B is 1,2,4-oxadiazole Cis selected from the group consisting of:

Also within this embodiment is encompassed the compound of Formula Iwherein B is 1,2,4-oxadiazole and C is thiophene or furan.

Another embodiment of the invention encompassed the compound of FormulaI wherein m=0 and A is —CO₂H. Within this embodiment is encompassed thecompound of Formula I wherein R¹, R² and R³ are hydrogen.

Another embodiment of the invention encompassed the compound of FormulaI wherein the group —B—C is attached to the phenyl ring at the4-position.

The invention also encompasses a compound represented by Formula II

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

-   n=0 or 1;-   R³ is selected from the group consisting of: hydrogen and C₁₋₄alkyl,    optionally substituted with from one up to the maximum number of    substitutable positions with a substituent independently selected    from the group consisting of: halo and hydroxy;-   each R⁴ is independently selected from the group consisting of:    halo, C₁₋₄alkyl and C₁₋₃alkoxy, said C₁₋₄alkyl and C₁₋₃alkoxy    optionally substituted from one up to the maximum number of    substitutable positions with halo.

Another embodiment of the invention encompassed a compound of Formula IIwherein n is 0.

Another embodiment of the invention encompassed a compound of Formula IIwherein n is 1.

Another embodiment of the invention encompassed a compound of Formula IIwherein R³ is hydrogen.

The invention also encompasses a compound represented by Formula III

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

-   n=0 or 1;-   R³ is selected from the group consisting of: hydrogen and C₁₋₄alkyl,    optionally substituted with from one up to the maximum number of    substitutable positions with a substituent independently selected    from the group consisting of: halo and hydroxy;-   each R⁴ is independently selected from the group consisting of:    halo, C₁₋₄alkyl and C₁₋₃alkoxy, said C₁₋₄alkyl and C₁₋₃alkoxy    optionally substituted from one up to the maximum number of    substitutable positions with halo.

Another embodiment of the invention encompassed a compound of FormulaIII wherein n is 0.

Another embodiment of the invention encompassed a compound of FormulaIII wherein n is 1.

Another embodiment of the invention encompassed a compound of Formula IIwherein R³ is hydrogen.

The invention also encompasses a method of treating an immunoregulatoryabnormality in a mammalian patient in need of such treatment comprisingadministering to said patient a compound of Formula I in an amount thatis effective for treating said immunoregulatory abnormality.

Within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is an autoimmune or chronic inflammatorydisease selected from the group consisting of: systemic lupuserythematosis, chronic rheumatoid arthritis, type I diabetes mellitus,inflammatory bowel disease, biliary cirrhosis, uveitis, multiplesclerosis, Crohn's disease, ulcerative colitis, bullous pemphigoid,sarcoidosis, psoriasis, autoimmune myositis, Wegener's granulomatosis,ichthyosis, Graves ophthalmopathy and asthma.

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is bone marrow or organ transplantrejection or graft-versus-host disease.

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is selected from the group consisting of:transplantation of organs or tissue, graft-versus-host diseases broughtabout by transplantation, autoimmune syndromes including rheumatoidarthritis, systemic lupus erythematosus, Hashimoto's thyroiditis,multiple sclerosis, myasthenia gravis, type I diabetes, uveitis,posterior uveitis, allergic encephalomyelitis, glomerulonephritis,post-infectious autoimmune diseases including rheumatic fever andpost-infectious glomerulonephritis, inflammatory and hyperproliferativeskin diseases, psoriasis, atopic dermatitis, contact dermatitis,eczematous dermatitis, seborrhoeic dermatitis, lichen planus, pemphigus,bullous pemphigoid, epidermolysis bullosa, urticaria, angioedemas,vasculitis, erythema, cutaneous eosinophilia, lupus erythematosus, acne,alopecia areata, keratoconjunctivitis, vernal conjunctivitis, uveitisassociated with Behcet's disease, keratitis, herpetic keratitis, conicalcornea, dystrophia epithelialis corneae, corneal leukoma, ocularpemphigus, Mooren's ulcer, scleritis, Graves' opthalmopathy,Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollen allergies, reversibleobstructive airway disease, bronchial asthma, allergic asthma, intrinsicasthma, extrinsic asthma, dust asthma, chronic or inveterate asthma,late asthma and airway hyper-responsiveness, bronchitis, gastric ulcers,vascular damage caused by ischemic diseases and thrombosis, ischemicbowel diseases, inflammatory bowel diseases, necrotizing enterocolitis,intestinal lesions associated with thermal burns, coeliac diseases,proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease,ulcerative colitis, migraine, rhinitis, eczema, interstitial nephritis,Goodpasture's syndrome, hemolytic-uremic syndrome, diabetic nephropathy,multiple myositis, Guillain-Barre syndrome, Meniere's disease,polyneuritis, multiple neuritis, mononeuritis, radiculopathy,hyperthyroidism, Basedow's disease, pure red cell aplasia, aplasticanemia, hypoplastic anemia, idiopathic thrombocytopenic purpura,autoimmune hemolytic anemia, agranulocytosis, pernicious anemia,megaloblastic anemia, anerythroplasia, osteoporosis, sarcoidosis,fibroid lung, idiopathic interstitial pneumonia, dermatomyositis,leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity,cutaneous T cell lymphoma, arteriosclerosis, atherosclerosis, aortitissyndrome, polyarteritis nodosa, myocardosis, scleroderma, Wegener'sgranuloma, Sjogren's syndrome, adiposis, eosinophilic fascitis, lesionsof gingiva, periodontium, alveolar bone, substantia ossea dentis,glomerulonephritis, male pattern alopecia or alopecia senilis bypreventing epilation or providing hair germination and/or promoting hairgeneration and hair growth, muscular dystrophy, pyoderma and Sezary'ssyndrome, Addison's disease, ischemia-reperfusion injury of organs whichoccurs upon preservation, transplantation or ischemic disease,endotoxin-shock, pseudomembranous colitis, colitis caused by drug orradiation, ischemic acute renal insufficiency, chronic renalinsufficiency, toxinosis caused by lung-oxygen or drugs, lung cancer,pulmonary emphysema, cataracta, siderosis, retinitis pigmentosa, senilemacular degeneration, vitreal scarring, corneal alkali burn, dermatitiserythema multiforme, linear IgA ballous dermatitis and cementdermatitis, gingivitis, periodontitis, sepsis, pancreatitis, diseasescaused by environmental pollution, aging, carcinogenesis, metastasis ofcarcinoma and hypobaropathy, disease caused by histamine orleukotriene-C₄ release, Behcet's disease, autoimmune hepatitis, primarybiliary cirrhosis, sclerosing cholangitis, partial liver resection,acute liver necrosis, necrosis caused by toxin, viral hepatitis, shock,or anoxia, B-virus hepatitis, non-A/non-B hepatitis, cirrhosis,alcoholic cirrhosis, hepatic failure, fulminant hepatic failure,late-onset hepatic failure, “acute-on-chronic” liver failure,augmentation of chemotherapeutic effect, cytomegalovirus infection, HCMVinfection, AIDS, cancer, senile dementia, trauma, and chronic bacterialinfection

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is multiple sclerosis

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is rheumatoid arthritis

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is systemic lupus erythematosus

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is psoriasis

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is rejection of transplanted organ ortissue

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is inflammatory bowel disease.

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is a malignancy of lymphoid originincluding acute and chronic lymphocytic leukemias and lymphomas.

The invention also encompasses a method of suppressing the immune systemin a mammalian patient in need of immunosuppression comprisingadministering to said patient an immunosuppressing effective amount of acompound of Formula I.

The invention also encompasses a pharmaceutical composition comprised ofa compound of Formula I in combination with a pharmaceuticallyacceptable carrier.

Exemplifying the invention are the following compounds:

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The invention is described using the following definitions unlessotherwise indicated.

The term “halogen” or “halo” includes F, Cl, Br, and I.

The term “alkyl” means linear or branched structures and combinationsthereof, having the indicated number of carbon atoms. Thus, for example,C₁₋₆alkyl includes methyl, ethyl, propyl, 2-propyl, s- and t-butyl,butyl, pentyl, hexyl, 1,1-dimethylethyl, cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

The term “alkoxy” means alkoxy groups of a straight, branched or cyclicconfiguration having the indicated number of carbon atoms. C₁₋₆alkoxy,for examples, includes methoxy, ethoxy, propoxy, isopropoxy, and thelike.

The term “alkylthio” means alkylthio groups having the indicated numberof carbon atoms of a straight, branched or cyclic configuration.C₁₋₆alkylthio, for example, includes methylthio, propylthio,isopropylthio, and the like.

The term “alkenyl” means linear or branched structures and combinationsthereof, of the indicated number of carbon atoms, having at least onecarbon-to-carbon double bond, wherein hydrogen may be replaced by anadditional carbon-to-carbon double bond. C₂₋₆alkenyl, for example,includes ethenyl, propenyl, 1-methylethenyl, butenyl and the like.

The term “alkynyl” means linear or branched structures and combinationsthereof, of the indicated number of carbon atoms, having at least onecarbon-to-carbon triple bond. C₃₋₆alkynyl, for example, includes ,propenyl, 1-methylethenyl, butenyl and the like.

The term “cycloalkyl” means mono-, bi- or tri-cyclic structures,optionally combined with linear or branched structures, the indicatednumber of carbon atoms. Examples of cycloalkyl groups includecyclopropyl, cyclopentyl, cycloheptyl, adamantyl, cyclododecylmethyl,2-ethyl-1-bicyclo[4.4.0]decyl, and the like.

The term “aryl” is defined as a mono- or bi-cyclic aromatic ring systemand includes, for example, phenyl, naphthyl, and the like.

The term “aralkyl” means an alkyl group as defined above of 1 to 6carbon atoms with an aryl group as defined above substituted for one ofthe alkyl hydrogen atoms, for example, benzyl and the like.

The term “aryloxy” means an aryl group as defined above attached to amolecule by an oxygen atom (aryl-O) and includes, for example, phenoxy,naphthoxy and the like.

The term “aralkoxy” means an aralkyl group as defined above attached toa molecule by an oxygen atom (aralkyl-O) and includes, for example,benzyloxy, and the like.

The term “arylthio” is defined as an aryl group as defined aboveattached to a molecule by an sulfur atom (aryl-S) and includes, forexample, thiophenyoxy, thionaphthoxy and the like.

The term “aroyl” means an aryl group as defined above attached to amolecule by an carbonyl group (aryl-C(O)—) and includes, for example,benzoyl, naphthoyl and the like.

The term “aroyloxy” means an aroyl group as defined above attached to amolecule by an oxygen atom (aroyl-O) and includes, for example,benzoyloxy or benzoxy, naphthoyloxy and the like.

The term “HET” is defined as a 5- to 10-membered aromatic, partiallyaromatic or non-aromatic mono- or bicyclic ring, containing 1-5heteroatoms selected from O, S and N, and optionally substituted with1-2 oxo groups. Preferably, “HET” is a 5- or 6-membered aromatic ornon-aromatic monocyclic ring containing 1-3 heteroatoms selected from O,S and N, for example, pyridine, pyrimidine, pyridazine, furan,thiophene, thiazole, oxazole, isooxazole and the like, or heterocycle isa 9- or 10-membered aromatic or partially aromatic-bicyclic ringcontaining 1-3 heteroatoms selected from O, S, and N, for example,benzofuran, benzothiophene, indole, pyranopyrrole, benzopyran,quionoline, benzocyclohexyl, naphtyridine and the like. “HET” alsoincludes the following: benzimidazolyl, benzofuranyl, benzopyrazolyl,benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl,cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl,indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl,isoxazolyl, naphthyridinyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl,pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl,quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thiazolyl, thienyl,triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl,piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl. Apreferred group of HET is as follows:

The term “treating” encompasses not only treating a patient to relievethe patient of the signs and symptoms of the disease or condition butalso prophylactically treating an asymptomatic patient to prevent theonset or progression of the disease or condition. The term “amounteffective for treating” is intended to mean that amount of a drug orpharmaceutical agent that will elicit the biological or medical responseof a tissue, a system, animal or human that is being sought by aresearcher, veterinarian, medical doctor or other clinician. The termalso encompasses the amount of a pharmaceutical drug that will preventor reduce the risk of occurrence of the biological or medical event thatis sought to be prevented in a tissue, a system, animal or human by aresearcher, veterinarian, medical doctor or other clinician.

The invention described herein includes pharmaceutically acceptablesalts and hydrates. Pharmaceutically acceptable salts include both themetallic (inorganic) salts and organic salts; a list of which is givenin Remington's Pharmaceutical Sciences, 17th Edition, pg. 1418 (1985).It is well known to one skilled in the art that an appropriate salt formis chosen based on physical and chemical stability, flowability,hydroscopicity and solubility. As will be understood by those skilled inthe art, pharmaceutically acceptable salts include, but are not limitedto salts of inorganic acids such as hydrochloride, sulfate, phosphate,diphosphate, hydrobromide, and nitrate or salts of an organic acid suchas malate, maleate, fumarate, tartrate, succinate, citrate, acetate,lactate, methanesulfonate, p-toluenesulfonate or pamoate, salicylate andstearate. Similarly pharmaceutically acceptable cations include, but arenot limited to sodium, potassium, calcium, aluminum, lithium andammonium (especially ammonium salts with secondary amines). Preferredsalts of this invention for the reasons cited above include potassium,sodium, calcium and ammonium salts. Also included within the scope ofthis invention are crystal forms, hydrates and solvates of the compoundsof Formula I.

For purposes of this Specification, “pharmaceutically acceptablehydrate” means the compounds of the instant invention crystallized withone or more molecules of water to form a hydrated form.

The invention also includes the compounds falling within formula I inthe form of one or more stereoisomers, in substantially pure form or inthe form of a mixture of stereoisomers. All such isomers are encompassedwithin the present invention.

By virtue of their S1P₁/Edg1 agonist activity, the compounds of thepresent invention are immunoregulatory agents useful for treating orpreventing automimmune or chronic inflammatory diseases. The compoundsof the present invention are useful to suppress the immune system ininstances where immunosuppression is in order, such as in bone marrow,organ or transplant rejection, autoimmune and chronic inflammatorydiseases, including systemic lupus erythematosis, chronic rheumatoidarthritis, type I diabetes mellitus, inflammatory bowel disease, biliarycirrhosis, uveitis, multiple sclerosis, Crohn's disease, ulcerativecolitis, bullous pemphigoid, sarcoidosis, psoriasis, autoimmunemyositis, Wegener's granulomatosis, ichthyosis, Graves ophthalmopathyand asthma.

More particularly, the compounds of the present invention are useful totreat or prevent a disease or disorder selected from the groupconsisting of: transplantation of organs or tissue, graft-versus-hostdiseases brought about by transplantation, autoimmune syndromesincluding rheumatoid arthritis, systemic lupus erythematosus,Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type Idiabetes, uveitis, posterior uveitis, allergic encephalomyelitis,glomerulonephritis, post-infectious autoimmune diseases includingrheumatic fever and post-infectious glomerulonephritis, inflammatory andhyperproliferative skin diseases, psoriasis, atopic dermatitis, contactdermatitis, eczematous dermatitis, seborrhoeic dermatitis, lichenplanus, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria,angioedemas, vasculitis, erythema, cutaneous eosinophilia, lupuserythematosus, acne, alopecia areata, keratoconjunctivitis, vernalconjunctivitis, uveitis associated with Behcet's disease, keratitis,herpetic keratitis, conical cornea, dystrophia epithelialis corneae,corneal leukoma, ocular pemphigus, Mooren's ulcer, scleritis, Graves'opthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollenallergies, reversible obstructive airway disease, bronchial asthma,allergic asthma, intrinsic asthma, extrinsic asthma, dust asthma,chronic or inveterate asthma, late asthma and airwayhyper-responsiveness, bronchitis, gastric ulcers, vascular damage causedby ischemic diseases and thrombosis, ischemic bowel diseases,inflammatory bowel diseases, necrotizing enterocolitis, intestinallesions associated with thermal burns, coeliac diseases, proctitis,eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerativecolitis, migraine, rhinitis, eczema, interstitial nephritis,Goodpasture's syndrome, hemolytic-uremic syndrome, diabetic nephropathy,multiple myositis, Guillain-Barre syndrome, Meniere's disease,polyneuritis, multiple neuritis, mononeuritis, radiculopathy,hyperthyroidism, Basedow's disease, pure red cell aplasia, aplasticanemia, hypoplastic anemia, idiopathic thrombocytopenic purpura,autoimmune hemolytic anemia, agranulocytosis, pernicious anemia,megaloblastic anemia, anerythroplasia, osteoporosis, sarcoidosis,fibroid lung, idiopathic interstitial pneumonia, dermatomyositis,leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity,cutaneous T cell lymphoma, arteriosclerosis, atherosclerosis, aortitissyndrome, polyarteritis nodosa, myocardosis, scleroderma, Wegener'sgranuloma, Sjogren's syndrome, adiposis, eosinophilic fascitis, lesionsof gingiva, periodontium, alveolar bone, substantia ossea dentis,glomerulonephritis, male pattern alopecia or alopecia senilis bypreventing epilation or providing hair germination and/or promoting hairgeneration and hair growth, muscular dystrophy, pyoderma and Sezary'ssyndrome, Addison's disease, ischemia-reperfusion injury of organs whichoccurs upon preservation, transplantation or ischemic disease,endotoxin-shock, pseudomembranous colitis, colitis caused by drug orradiation, ischemic acute renal insufficiency, chronic renalinsufficiency, toxinosis caused by lung-oxygen or drugs, lung cancer,pulmonary emphysema, cataracta, siderosis, retinitis pigmentosa, senilemacular degeneration, vitreal scarring, corneal alkali burn, dermatitiserythema multiforme, linear IgA ballous dermatitis and cementdermatitis, gingivitis, periodontitis, sepsis, pancreatitis, diseasescaused by environmental pollution, aging, carcinogenesis, metastasis ofcarcinoma and hypobaropathy, disease caused by histamine orleukotriene-C₄ release, Behcet's disease, autoimmune hepatitis, primarybiliary cirrhosis, sclerosing cholangitis, partial liver resection,acute liver necrosis, necrosis caused by toxin, viral hepatitis, shock,or anoxia, B-virus hepatitis, non-A/non-B hepatitis, cirrhosis,alcoholic cirrhosis, hepatic failure, fulminant hepatic failure,late-onset hepatic failure, “acute-on-chronic” liver failure,augmentation of chemotherapeutic effect, cytomegalovirus infection, HCMVinfection, AIDS, cancer, senile dementia, trauma, and chronic bacterialinfection.

Also embodied within the present invention is a method of preventing ortreating resistance to transplantation or transplantation rejection oforgans or tissues in a mammalian patient in need thereof, whichcomprises administering a therapeutically effective amount of thecompound of Formula I.

A method of suppressing the immune system in a mammalian patient in needthereof, which comprises administering to the patient an immune systemsuppressing amount of the compound of Formula I is yet anotherembodiment.

Most particularly, the method described herein encompasses a method oftreating or preventing bone marrow or organ transplant rejection whichis comprised of administering to a mammalian patient in need of suchtreatment or prevention a compound of formula I, or a pharmaceuticallyacceptable salt or hydrate thereof, in an amount that is effective fortreating or preventing bone marrow or organ transplant rejection.

Furthermore, a preferred group of compounds of the present invention areagonists of the S1P₁/Edg1 receptor having selectivity over S1P₃/Edg3receptor. An Edg1 selective agonist has advantages over currenttherapies and extends the therapeutic window of lymphocytessequestration agents, allowing better tolerability with higher dosingand thus improving efficacy as monotherapy. The following compoundspossesses a selectivity for the S1P₁/Edg1 receptor over the S1PR₃/Edg3receptor of at least 20 fold as measured by the ratio of EC₅₀ for theS1P₁/Edg1 receptor to the EC₅₀ for the S1P₃/Edg3 receptor as evaluatedin the ³⁵S-GTPγS binding assay and possesses an EC₅₀ for binding to theS1P₁/Edg1 receptor of 100 nM or less as evaluated by the ³⁵S-GTPγSbinding assay:

The present invention also includes a pharmaceutical formulationcomprising a pharmaceutically acceptable carrier and the compound ofFormula I or a pharmaceutically acceptable salt or hydrate thereof. Apreferred embodiment of the formulation is one where a secondimmunosuppressive agent is also included. Examples of such secondimmunosuppressive agents are, but are not limited to azathioprine,brequinar sodium, deoxyspergualin, mizaribine, mycophenolic acidmorpholino ester, cyclosporin, FK-506, rapamycin and FTY720.

The present compounds, including salts and hydrates thereof, are usefulin the treatment of autoimmune diseases, including the prevention ofrejection of bone marrow transplant, foreign organ transplants and/orrelated afflictions, diseases and illnesses.

The compounds of this invention can be administered by any means thateffects contact of the active ingredient compound with the site ofaction in the body of a warm-blooded animal. For example,administration, can be oral, topical, including transdermal, ocular,buccal, intranasal, inhalation, intravaginal, rectal, intracisternal andparenteral. The term “parenteral” as used herein refers to modes ofadministration which include subcutaneous, intravenous, intramuscular,intraarticular injection or infusion, intrasternal and intraperitoneal.

The compounds can be administered by any conventional means availablefor use in conjunction with pharmaceuticals, either as individualtherapeutic agents or in a combination of therapeutic agents. They canbe administered alone, but are generally administered with apharmaceutical carrier selected on the basis of the chosen route ofadministration and standard pharmaceutical practice.

The dosage administered will be dependent on the age, health and weightof the recipient, the extent of disease, kind of concurrent treatment,if any, frequency of treatment and the nature of the effect desired.Usually, a daily dosage of active ingredient compound will be from about0.1-2000 milligrams per day. Ordinarily, from 1 to 100 milligrams perday in one or more applications is effective to obtain desired results.These dosages are the effective amounts for the treatment of autoimmunediseases, the prevention of rejection of foreign organ transplantsand/or related afflictions, diseases and illnesses.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, troches, dragées, granules and powders, or inliquid dosage forms, such as elixirs, syrups, emulsions, dispersions,and suspensions. The active ingredient can also be administeredparenterally, in sterile liquid dosage forms, such as dispersions,suspensions or solutions. Other dosages forms that can also be used toadminister the active ingredient as an ointment, cream, drops,transdermal patch or powder for topical administration, as an ophthalmicsolution or suspension formation, i.e., eye drops, for ocularadministration, as an aerosol spray or powder composition for inhalationor intranasal administration, or as a cream, ointment, spray orsuppository for rectal or vaginal administration.

Gelatin capsules contain the active ingredient and powdered carriers,such as lactose, starch, cellulose derivatives, magnesium stearate,stearic acid, and the like. Similar diluents can be used to makecompressed tablets. Both tablets and capsules can be manufactured assustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose),and related sugar solutions and glycols such as propylene glycol orpolyethylene gycols are suitable carriers for parenteral solutions.Solutions for parenteral administration preferably contain a watersoluble salt of the active ingredient, suitable stabilizing agents, andif necessary, buffer substances. Antioxidizing agents such as sodiumbisulfite, sodium sulfite, or ascorbic acid, either alone or combined,are suitable stabilizing agents. Also used are citric acid and its saltsand sodium EDTA. In addition, parenteral solutions can containpreservatives, such as benzalkonium chloride, methyl- or propylparaben,and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, A. Osol, a standard reference text in thisfield.

For administration by inhalation, the compounds of the present inventionmay be conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or nebulisers. The compounds mayalso be delivered as powders which may be formulated and the powdercomposition may be inhaled with the aid of an insufflation powderinhaler device. The preferred delivery system for inhalation is ametered dose inhalation (MDI) aerosol, which may be formulated as asuspension or solution of a compound of Formula I in suitablepropellants, such as fluorocarbons or hydrocarbons.

For ocular administration, an ophthalmic preparation may be formulatedwith an appropriate weight percent solution or suspension of thecompounds of Formula I in an appropriate ophthalmic vehicle, such thatthe compound is maintained in contact with the ocular surface for asufficient time period to allow the compound to penetrate the cornealand internal regions of the eye.

Useful pharmaceutical dosage-forms for administration of the compoundsof this invention can be illustrated as follows:

Capsules

A large number of unit capsules are prepared by filling standardtwo-piece hard gelatin capsules each with 100 milligrams of powderedactive ingredient, 150 milligrams of lactose, 50 milligrams ofcellulose, and 6 milligrams magnesium stearate.

Soft Gelatin Capsules

A mixture of active ingredient in a digestible oil such as soybean oil,cottonseed oil or olive oil is prepared and injected by means of apositive displacement pump into gelatin to form soft gelatin capsulescontaining 100 milligrams of the active ingredient. The capsules arewashed and dried.

Tablets

A large number of tablets are prepared by conventional procedures sothat the dosage unit is 100 milligrams of active ingredient, 0.2milligrams of colloidal silicon dioxide, 5 milligrams of magnesiumstearate, 275 milligrams of microcrystalline cellulose, 11 milligrams ofstarch and 98.8 milligrams of lactose. Appropriate coatings may beapplied to increase palatability or delay absorption.

Injectable

A parenteral composition suitable for administration by injection isprepared by stirring 1.5% by weight of active ingredient in 10% byvolume propylene glycol. The solution is made to volume with water forinjection and sterilized.

Suspension

An aqueous suspension is prepared for oral administration so that each 5milliliters contain 100 milligrams of finely divided active ingredient,100 milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodiumbenzoate, 1.0 grams of sorbitol solution, U.S.P., and 0.025 millilitersof vanillin.

The same dosage forms can generally be used when the compounds of thisinvention are administered stepwise or in conjunction with anothertherapeutic agent. When drugs are administered in physical combination,the dosage form and administration route should be selected depending onthe compatibility of the combined drugs. Thus the term coadministrationis understood to include the administration of the two agentsconcomitantly or sequentially, or alternatively as a fixed dosecombination of the two active components.

Methods of Synthesis

Two general methods that can be employed to prepare compounds in thecurrent invention are depicted in Scheme 1. Intermediates i may beavailable from commercial sources (e.g., azetidine-3-carboxylic acid,where R₁=H, R₂=H, m=0, n=0 or pyrrolidine-3-carboxylic acid, where R₁=H,R₂=H, m=0, n=1) or they can be prepared using methods described below.Combining i with an aryl aldehyde ii in the presence of an appropriatereducing agent (e.g., sodium cyanoborohydride, sodiumtriacetoxyborohydride, sodium borohydride) in a compatible solvent(e.g., methanol, ethanol, acetonitrile, methylene chloride) can affordcompounds of structure iii. Alternatively, intermediates i can becombined with a benzyl halide or sulfonate ester iv in the presence ofan appropriate base (e.g., sodium carbonate, potassium carbonate,triethylamine, N,N-diisopropylethylamine) in a compatible solventsolvent (e.g., methanol, ethanol, acetonitrile) at or above roomtemperature to give compounds of structure iii. In cases where A instructure i would interfere with the transformation to iii, anappropriate protecting group (Greene & Wuts, eds., “Protecting Groups inOrganic Synthesis”, John Wiley & Sons, Inc.) that would mask A and allowfor the liberation of A after coupling with either ii or iv can beemployed. In cases where iii contains asymmetric centers, the individualstereoisomers of iii can obtained by methods known to those skilled inthe art which include (but are not limited to): stereospecificsynthesis, resolution of salts of iii or any of the intermediates usedin its preparation with enantiopure acids or bases, resolution of iii orany of the intermediates used in its preparation by HPLC employingenantiopure stationary phases.

Compounds in the current invention in which m=0, n=1 and A=—CO₂H can beprepared using methods shown in Scheme 2. An acrylic acid (v)substituted with

functional groups R₁ and/or R₂ (e.g., R₁ and/or R₂=H, alkyl,trihaloalkyl or carboxy) can be reacted with an azomethine ylidegenerated from vi in the presence of a catalytic amount of an acid(e.g., trifluoroacetic acid, phosphoric acid) in an appropriate solvent(e.g., methylene chloride, acetonitrile) to give compounds of thestructure vii. Alternatively, viii (prepared similarly to vii, butemploying an acrylate ester as the starting material) can be treatedwith a strong base (e.g., lithium diisopropylamide, sodiumbis(trimethylsilyl)amide, potassium bis(trimethylsilyl)amide) in anethereal solvent (e.g., THF, 1,2-dimethoxyethane) at or below roomtemperature followed by an electrophile (e.g., methyl iodide,2-(phenylsulfonyl)-3-phenyloxaziridine, fluorobenzenesulfonimide) togive ix. Saponification of ix can then give vii. In cases

where vii contains asymmetric centers, individual stereoisomers can beobtained using methods similar to those described for iii in Scheme 1.

Several methods that can be used to prepare compounds that could beemployed as intermediate i in Scheme 1 above are shown in Scheme 3. Forcases where m=0, n=1, R₁=H, R₂=H and A=—PO₃H₂, diethyl vinylphosphonate(x) can be reacted with N-methoxymethyl-N-trimethylsilylmethyl benzylamine in in the presence of a catalytic amount of an acid (e.g.,trifluoroacetic acid, phosphoric acid) in an appropriate solvent (e.g.,methylene chloride, acetonitrile) to a give compound of the structurexi. Cleavage of the N-benzyl group using catalytic hydrogenation (H₂,Pd(OH)₂/C, HOAc; ammonium formate, Pd(OH)₂/C, MeOH) or chemical methods(1-chloroethyl chloroformate, DCE, reflux, followed by MeOH, reflux) cangive xii. For cases where m=0, n=1, R₁=OH, R₂=H and A=—PO₃H₂,N-t-butoxycarbonyl protection of 3-hydroxypyrrolidine (xiii) followed bymild oxidation (e.g., treatment with oxalyl chloride and DMSO at −78° C.in dichloromethane followed by a trialkylamine base and warming (Swernoxidation); treatment with 4-methylmorpholine N-oxide and catalytictetrapropylammonium peruthenate in acetonitrile) can give xiv. Treatingxiv with a dialkylphosphite in the presence of a tertiary amine base(triethylamine, N,N-diisopropylethylamine) at or above room temperaturefollowed by removal of the t-butylcarbamate under acidic conditions(e.g., HCl in MeOH, neat TFA) can give xv. For cases where m=0, n=1,R₁=H, R₂=H and A=5-tetrazolyl, acrylonitrile (xvi) can be reacted withN-methoxymethyl-N-trisilylmethyl benzyl amine in the presence of acatalytic amount of an acid (e.g., trifluoroacetic acid, phosphoricacid) in an appropriate solvent (e.g., methylene chloride, acetonitrile)to a give compound of the structure xvii. Converting the N-benzyl groupof xvii to a benzyl carbamate following by tetrazole formation (e.g.,ammonium chloride, sodium azide, DMF at elevated temperature;trimethyltin azide, toluene, reflux) then catalytic hydrogenation cangive xviii.

Several methods that can be used to prepare compounds that can beemployed as intermediate ii in Scheme 1 above are shown in Scheme 4.Many aryl carboxylic acids, aryl carboxylic acid halides, arylcarboxylic esters, and aryl N-alkoxyl-N-alkyl carboxamides (xix) arecommercially available and can be converted to aryl aldehydes (xx) usingreduction methods known by those skilled in the art (see Larock,“Comprehensive Organic Transformations, A Guide to Functional GroupPreparations”, VCH Publishers, Inc.). Alternatively, many benzylalcohols (xxi) are commercially

available and can be converted to aryl aldehydes (xxii) using oxidationmethods known by those skilled in the art. For cases where B=alkoxy, ahydroxy benzaldehyde xxi can be combined with a alkyl halide orsulfonate ester in the presence of an appropriate base (e.g., sodiumcarbonate, potassium carbonate, triethylamine,N,N-diisopropylethylamine) in a compatible solvent solvent (e.g.,methanol, ethanol, acetonitrile) at or above room temperature to givecompounds of structure xxiv. Alternatively, a hydroxy benzaldehyde xxiiican be combined with an alcohol, a dialkyl azodicarboxylate (e.g.,diethyl azodicarboxylate, diisopropylazodicarboxylate) andtriphenylphosphine in an appropriate solvent (THF, toluene, methylenechloride) to give xxiv. For cases where B is 1,2,4-oxadiazolyl,N-hydroxyamidine xxv can be treated with an acid chloride in anappropriate solvent (xylenes, toluene) in the presence of an amine base(pyridine, DBU) with heating to give an intermediate xxvi.Alternatively, xxv can be treated with a carboxylic acid, a carbodiimide(e.g., N,N′-dicyclohexylcarbodiimide,1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and1-hydroxybenzotriazole in an appropriate solvent (xylenes, toluene) togive xxvi. Prepared by either manner, the ester group of xxvi can beconverted to aldehyde with methods employed to convert xix to xx. Forcases where B is —(C═O)C₆₋₁₁ alkyl and R₄=H, an aryl 1,4-dialdehyde(xxvii) can be treated with a limiting amount of an alkyl organometallicreagent (e.g., alkyl magnesium bromide, alkyl lithium) at or below roomtemperature in an ethereal solvent (e.g., THF, diethyl ether,1,2-dimethoxyethane) to afford intermediate xxviii. Mild oxidation ofxxviii (e.g., treatment with oxalyl chloride and DMSO at −78° C. indichloromethane followed by a trialkylamine base and warming (Swernoxidation); treatment with 4-methylmorpholine N-oxide and catalytictetrapropylammonium peruthenate in acetonitrile) can give aldehyde xix.

Methods for preparing the compounds of this invention are furtherillustrated in the following examples. Alternative routes will be easilydiscernible to practitioners in the field.

General

Concentration of solutions was carried out on a rotary evaporator underreduced pressure. Conventional flash chromatography was carried out onsilica gel (230-400 mesh). Flash chromatography was also carried outusing a Biotage Flash Chromatography apparatus (Dyax Corp.) on silicagel (32-63 mM, 60 Å pore size) in pre-packed cartridges of the sizenoted. NMR spectra were obtained in CDCl₃ solution unless otherwisenoted. Coupling constants (J) are in hertz (Hz). Abbreviations: diethylether (ether), triethylamine (TEA), N,N-diisopropylethylaamine (DIEA),saturated aqueous (sat'd), room temperature (rt), hour(s) (h or hr),minute(s) (min). For the tables that follow any NMR data follows thecompounds.

HPLC Conditions

LC-1: Waters Xterra MS C18, 5μ, 4.6×50 mm column, 10:90 to 95:5 v/vCH₃CN/H₂O+0.05% TFA over 4.5 min, hold 1 min, PDA detection 200-600 nm,flow rate=2.5 mL/min.

LC-2: Analytical Sales and Service Armor C8 5μ 20×100 mm column, 10:90to 90:10 v/v CH₃CN/H₂O+0.05% TFA over 12 min, hold 4 min, UV detectionat either 210 or 254 nM, flow rate=10 mL/min.

Preparation of Aldehyde Intermediates

Aldehyde 1

4-Nonylbenzaldehyde

A solution of 2.0 g (7.5 mmol) of 4-nonylbenzoyl chloride in 75 mL ofTHF at −78° C. was treated with 7.5 mL (7.5 mmol) of 1M lithiumtri-(tert-butoxy) aluminum hydride in THF. After 30 min at −78° C., thereaction was quenched with 2N HCl and was allowed to warm to rt. Themixture was poured into Et₂O and washed with 2N HCl, sat'd NaHCO₃ andsat'd NaCl. The organic layer was dried over MgSO₄ and concentrated. Theresidue was purified on a 40M Biotage column using 100:1 v/v hexane/Et₂Oas the eluant to afford 708 mg (41%) of the title compound: ¹H-NMR (500MHz) δ 0.87 (t, J=7.0, 3H), 1.26-1.31 (m, 12H), 1.60-1.66 (m, 2H), 2.68(t, J=7.8, 2H), 7.32 (d, J=8.0, 2H), 7.79 (d, J=8.0, 2H), 9.97 (s, 1H).

Aldehyde 2

4-Decylbenzaldehyde

The title compound was prepared using a procedure analogous to Aldehyde1 substituting 4-decylbenzoyl chloride for 4-nonylbenzoyl chloride:¹H-NMR (500 MHz) δ 0.87 (t, J=6.9, 3H, 1.25-1.31 (m, 14H), 1.60-1.66 (m,2), 2.68 (t, J=7.7, 2H), 7.33 (d, J=8.0, 2H), 7.79 (d, J=8.0, 2H), 9.97(s, 1H).

Aldehyde 3

3-(Octyloxy)benzaldehyde

A mixture of 1.00 g (0.82 mmol) of 3-hydroxybenzaldehyde, 1.70 g (12.2mmol) of potassium carbonate and 2.16 g (9.00 mmol) of 1-iodooctane werewarmed in acetonitrile at 80° C. for 16 h. The reaction was cooled,filtered and concentrated. The residue was purified using flashchromatography using 20:1 v/v hexane/ethyl acetate to afford 1.63 g ofthe title compound as a colorless oil: ¹H-NMR (500 MHz) δ 0.89 (t,J=6.9, 3H), 1.24-1.39 (m, 8H), 1.42-1.50 (m, 2H), 1.80 (m, 2H), 4.01 (t,J=6.6, 2H), 7.19 (m, 1H), 7.40 (s, 1H), 7.44-7.46 (m, 2H), 9.99 (s, 1H).

Aldehyde 4

4-(Octyloxy)benzaldehyde

The title compound was prepared using a procedure analogous to Aldehyde3 substituting 4-hydroxybenzaldehyde for 3-hydroxybenzaldehyde: ¹H NMR(500 MHz) δ 0.91 (t, J=6.9, 3H), 1.29-1.41 (m, 8H), 1.46-1.52 (m, 2H),1.71-1.86 (m, 2H), 4.06 (t, J=6.6, 2H), 7.01 (d, J=8.7, 2H), 7.85 (d,J=8.7, 2H), 9.90 (s, 1H).

Aldehyde 5

3-Bromo-5-methoxy-4-octyloxybenzaldehyde

The title compound was prepared using a procedure analogous to Aldehyde3 substituting 3-bromo-4-hydroxy-5-methoxybenzaldehyde for3-hydroxybenzaldehyde: ESI-MS: 343 (M+H)

Aldehyde 6

3-Ethoxy-4-(octyloxy)benzaldehyde

The title compound was prepared using a procedure analogous to Aldehyde3 substituting 3-ethoxy-4-hydroxybenzaldehyde for 3-hydroxybenzaldehyde:¹H-NMR (500 MHz) δ 0.88-0.98 (m, 3H), 1.30-1.41 (m, 8H), 1.46-1.51 (m,5H), 1.85-1.91 (m, 2H), 4.06-4.18 (m, 4H), 6.97 (d, J=8.0, 1H),7.39-7.44 (m, 2H), 9.84 (s, 1H); ESI-MS 279.1 (M+H).

Aldehyde 7

3,5-Dibromo-4-(octyloxy)benzaldehyde

The title compound was prepared using a procedure analogous to Aldehyde3 substituting 3,5-dibromo-4-hydroxybenzaldehyde for3-hydroxybenzaldehyde.

Aldehyde 8

3-Methoxy-4-(octyloxy)benzaldehyde

The title compound was prepared using a procedure analogous to Aldehyde3 substituting 3-methoxy-4-hydroxybenzaldehyde for3-hydroxybenzaldehyde: ESI-MS 265.2 (M+H)

Aldehyde 9

3-Methyl-4-(octyloxy)benzaldehyde

The title compound was prepared using a procedure analogous to Aldehyde3 substituting 3-methyl-4-hydroxybenzaldehyde for 3-hydroxybenzaldehyde.

Aldehyde 10

4-(Octyloxy)-1-naphthaldehyde

The title compound was prepared using a procedure analogous to Aldehyde3 substituting 4-hydroxy-1-naphthaldehyde for 3-hydroxybenzaldehyde.

Aldehyde 11

2-Chloro-4-(octyloxy)benzaldehyde

The title compound was prepared using a procedure analogous to Aldehyde3 substituting 2-chloro-4-hydroxybenzaldehyde for 3-hydroxybenzaldehyde:ESI-MS 269.0 (M+H)

Aldehyde 12

3-Chloro-4-(octyloxy)benzaldehyde

The title compound was prepared using a procedure analogous to Aldehyde3 substituting 3-chloro-4-hydroxybenzaldehyde for 3-hydroxybenzaldehyde.

Aldehyde 13

4-(trans-3,7-Dimethyl-2,6-octadien-1-yloxy)benzaldehyde

The title compound was prepared using a procedure analogous to Aldehyde3 using 4-hydroxybenzaldehyde and geranyl bromide: R_(F): 0.29 (19:1 v/vhexane/EtOAc); ¹H-NMR (500 MHz) δ 1.58-1.83 (m, 9H), 2.00-2.16 (m, 4H),4.65 (d, J=6.6, 2H), 5.10 (m, 1H), 5.50 (m, 1H), 7.02 (d, J=8.7, 2H),7.85 (d, J=8.7, 2H), 9.90 (s, 1H).

Aldehyde 14

4-[Bis(3,5-trifluoromethyl)benzyloxy]benzaldehyde

The title compound was prepared using a procedure analogous to Aldehyde3 using 4-hydroxybenzaldehyde and bis(3,5-trifluoromethyl)benzylbromide: R_(F): 0.28 (9:1 v/v hexane/EtOAc); ¹H-NMR (500 MHz) δ 5.28 (s,2H), 7.14 (d, J=8.7, 2H), 7.91-7.95 (m, 5H), 9.95 (s, 1H).

Aldehyde 15

3-(4-(Formyl)phenyl)-5-(4-phenyl-5-trifluoromethyl-2-thienyl)-1,2,4-oxadiazole

Step A: (E/Z)-2-Phenyl-3-chloro-4,4,4-trifluoro-2-butanal

Phosphorous oxychloride (7.5 mL, 80 mmol) was added to 15 mL of DMF at0° C. The resulting mixture was warmed to rt and stirred for 1 h. Asolution of 5.0 g (26.6 mmol) of1,1,1-trifluoromethyl-3-phenyl-2-propanone in 1 mL of DMF was added andthe resulting mixture was stirred at 70° C. for 20 h. The reactionmixture was cooled to rt, poured onto 150 g of ice and stirred atambient temperature for 1 h. The quenched mixture was extracted with 200mL of ether. The extract was washed with 200 mL of water, dried andconcentrated. Chromatography on a Biotage 40 M cartridge using hexanes(4 L) as the eluant afforded 5.1 g (82%) of the title compound.

Step B: Ethyl (4-phenyl-5-trifluoromethyl)thiophene-2-carboxylate

Ethyl mercaptoacetate (2.75 mL, 25.0 mmol) was added to a suspension of600 mg (25 mmol) of NaH in 45 mL of THF maintaining the internaltemperature at 25° C. A solution of 5.10 g (21.7 mmol) of(E/Z)-2-phenyl-3-chloro-4,4,4-trifluoro-2-butanal (from Step A) wasadded and the resulting mixture was stirred at rt for 20 h. The reactionwas quenched with 50 mL of sat'd NH₄Cl and the resulting mixture waspartitioned between 250 mL of ether and 100 mL of water. The organiclayer was separated, dried and concentrated. Chromatography on a Biotage40 M cartridge using hexanes (1L), then 4:1 v/v hexanes/CH₂Cl₂ (1 L) asthe eluant afforded 5.10 g (78%) of the title compound: ¹H NMR (400 Mhz)δ 1.40 (t, J=7.2, 3H), 4.39 (q, J=7.2, 2H), 7.42 (app s, 5H), 7.74 (q,J=1.6, 1H).

Step C: (4-Phenyl-5-trifluoromethyl)thiophene-2-carboxylic acid

A solution of 5.10 g (17.0 mmol) of ethyl4-phenyl-5-trifluoromethyl-thiophene-2-carboxylate (from Step B) in 20mL of EtOH was treated with 10 mL of 5.0 N NaOH and stirred at rt for 30min. The EtOH was removed in vacuo. The residual aqueous mixture wasacidified to pH 2 with 1 N HCl, then extracted with 300 mL of 1:1 v/vEtOAc/ether. The extract was separated, dried and concentrated.Recrystallization from 200 mL of 20:1 v/v hexanes/ether afforded 4.30 g(93%) of the title compound: ¹H NMR (500 Mhz) δ 7.43 (app s, 5H), 7.84(app s, 1H); ¹³C NMR (CDCl₃, 125 Mhz) δ 121.7 (q, J=269), 128.5, 128.6,128.8, 132.5 (q, J=36), 133.3, 133.8, 137.5, 144.8, 167.0.

Step D:3-[4-(Carbomethoxy)phenyl]-5-(4-phenyl-5-trifluoromethyl-2-thienyl)-1,2,4-oxadiazole

A solution of 408 mg (1.5 mmol) of4-phenyl-5-trifluoromethyl-thiophene-2-carboxylic acid and 1 mL ofoxalyl chloride in 5 mL of CH₂Cl₂ was treated with 5 drops of DMF. Theresulting mixture was stirred at rt for 1 h, then concentrated. Thecrude acid chloride and 291 mg (1.5 mmol) of4-(carbomethoxy)benzamidoxime were dissolved in 7 mL of 6:1 v/vxylenes/pyridine. The resulting solution was heated at 140° C. for 1 h,then cooled. The mixture was partitioned between 50 mL of 1:1EtOAc/ether and 50 mL of 1 N HCl. The organic layer was separated,washed with 3×50 mL of 1 N HCl, 50 mL of sat'd NaHCO₃, dried andconcentrated. Chromatography on a Biotage 40 M cartridge using hexanes(1L), then 20:1 v/v hexanes/EtOAc (1 L) as the eluant afforded 423 mg(65%) of the title compound: ¹H NMR (500 Mhz) δ0 3.97 (s, 3H), 7.48 (apps, 5H), 7.92 (s, 1H), 8.18 (app d, J=8.5, 2H), 8.23 (app d, J=8.5, 2H).

Step E:3-[4-(Hydroxymethyl)phenyl]-5-(4-phenyl-5-trifluoromethyl-2-thienyl)-1,2,4-oxadiazole

A solution of 390 mg (0.91 mmol) of3-[4-(carbomethoxy)phenyl]-5-(4-phenyl-5-trifluoromethyl-2-thienyl)-1,2,4-oxadiazole(from Step D) in 10 mL of CH₂Cl₂ at −78° C. was treated with 2.7 mL of1.0 M DIBALH solution in CH₂Cl₂. The resulting solution was stirred coldfor 1 h, then quenched with 5 mL of sat'd Rochelle salt solution. Themixture was partitioned between 100 mL CH₂Cl₂ and 50 mL of 1 N NaOH. Theorganic layer was separated, dried and concentrated. Chromatography on aBiotage 40 S cartridge using 4:1 v/v hexanes/EtOAc (1L) as the eluantafforded 325 mg (89%) of the title compound: ¹H NMR (500 Mhz) δ 1.80(app s, 1H), 4.80 (d, J=4.0, 2H), 7.46-7.48 (5H), 7.52 (d, J=8.0, 2H),7.91 (q, J=1.5, 1H), 8.14 (d, J=8.0, 2H).

Step P:3-[4-(Formyl)phenyl]-5-(4-phenyl-5-trifluoromethyl-2-thienyl)-1,2,4-oxadiazole

A mixture of 310 mg (0.77 mmol) of3-[4-(hydroxymethyl)phenyl]-5-(4-phenyl-5-trifluoromethyl-2-thienyl)-1,2,4-oxadiazole(from Step E), 527 mg (1.5 mmol) of 4-methylmorpholine N-oxide and 500mg of 4 A molecular sieves in 15 mL of CH₃CN was treated with 12 mg(0.034 mmol) of tetrapropylammonium perruthnate and the resultingmixture was stirred ar rt for 2 h. The solids were filtered and thefiltrated was concentrated. Chromatography on a Biotage 40 S cartridgeusing 9:1 v/v hexanes/EtOAc (1L) as the eluant afforded 205 mg (66%) ofthe title compound: ¹H NMR (500 Mhz) δ 7.48 (app s, 5H), 7.93 (app s,1H), 8.03 (d, J=8.5, 2H), 8.33 (d, J=8.5, 2H), 10.1 (s, 1H).

Aldehyde 16

4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzaldehyde

Step A: 2-Hydroxymethyl-4-phenyl-5-trifluoromethyl-thiophene

A solution of 2.10 g (7.7 mmol) of4-phenyl-5-trifluoromethyl-thiophene-2-carboxylic acid (from Aldehyde15, Step C) in 20 mL of THF was treated with 5.0 mL of 2.0 M boranedimethylsulfide complex in THF. The resulting solution was heated atreflux for 3 h, cooled to rt, quenched with 10 mL of MeOH andconcentrated. Chromatography on a Biotage 40M cartridge using 9:1 v/vhexanes/EtOAc as the eluant afforded 1.95 g (98%) of the title compound:¹H NMR (500 Mhz) δ 2.05 (app s, 1H), 4.87 (s, 2H), 6.99 (s, 1H), 7.41(app s, 5H).

Step B: 4-((4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy)benzaldehyde

A solution of 1.95 g (7.5 mmol) of2-hydroxymethyl-4-phenyl-5-trifluoromethyl-thiophene (from Step A), 925mg (7.6 mmol) of 4-hydroxybenzaldehyde and 3.0 g (11.4 mmol) oftriphenylphosphene in 40 mL of THF at 0° C. was treated with 2.0 g (11.4mmol) of diethylazodicarboxylate. The resulting mixture was warmed tort, stirred for 2 h, then concentrated. Chromatography on a Biotage 75Scartridge using 9:1 v/v heptane/EtOAc as the eluant afforded 2.5 g ofimpure title compound. Chromatography on a Biotage 40M cartridge using19:1 v/v hexanes/EtOAc (1L), then 4:1 v/v hexanes/EtOAc (1L) as theeluant afforded 1.65 g (60%) of the title compound: ¹H NMR (500 Mhz) δ5.32 (s, 2H), 7.10 (d, J=8.5, 2H), 7.12 (s, 1H), 7.41-7.43 (5H),7.85-7.90 (2H), 9.92 (s, 1H).

Aldehydes 17-21 were prepared using procedures analogous to thosedescribed in Aldehyde 16 substituting the appropriately substitutedbenzaldehyde for 4-(hydroxy)benzaldehyde in Step B:

Aldehyde 17

3-((4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy)benzaldehyde

Aldehyde 18

2-Chloro-4-((4-phenyl-5-trifluoromethyl-2-thienyl)methoxy)benzaldehyde

Aldehyde 19

3-Chloro-4-((4-phenyl-5-trifluoromethyl-2-thienyl)methoxy)benzaldehyde

Aldehyde 20

3-Methyl-4-((4-phenyl-5-trifluoromethyl-2-thienyl)methoxy)benzaldehyde

Aldehyde 21

3-Methoxy-4-((4-phenyl-5-trifluoromethyl-2-thienyl)methoxy)benzaldehyde

Aldehyde 22

4-(4-Phenylbutoxy)benzaldehyde

The title compound was prepared using a procedure analogous to Aldehyde4 substituting 4-(iodobutyl)benzene for 1-iodooctane: ESI-MS 255.2 (M+H)

Aldehyde 23

4-(Non-1-oyl)benzaldehyde

Step A: 4-(1-Hydroxynon-1-yl)benzaldehyde

Terephthaldicarboxaldehyde (2.00 g, 14.91 mmol) was dissolved intetrahydrofuran (25 ml) and cooled to 0° C. Octylmagnesium chloride (7.5ml, 2.0M in THF, 15 mmol) was added dropwise. After 15 minutes, thereaction was quenched with 2N aqueous hydrochloric acid (50 ml) anddiluted with ethyl acetate (50 ml). The organic layer was separated,washed with sat'd NaCl (50 ml), dried over magnesium sulfate andconcentrated. Silica gel chromatography eluting with 91:9 v/vhexane/EtOAc gave 0.19 g (0.77 mmol, 5.1%) of the title compound: ¹H NMR(500 MHz) δ 10.0 (s, 1H), 7.87 (d, J=8.0, 2H), 7.52 (d, J=8.3, 2H),4.75-4.80 (m, 1H), 1.68-1.82 (m, 2H), 1.22-1.45 (m, 12H), 0.91 (t,J=7.0, 3H).

Step B: 4-(Non-1-oyl)benzaldehyde

Dess-Martin periodinane (0.268 g, 0.632 mmol) was added to a solution of4-(1-hydroxynon-1-yl)benzaldehyde (0.125 g, 0.505 mmol) from Step A inCH₂Cl₂ (3.0 ml). After 1 h, the reaction was filtered and concentrated.Silica gel chromatography eluting with 19:1 v/v hexane/EtOAc gave 0.107g (0.446 mmol, 88%) of the title compound: ¹H NMR (500 MHz) δ 10.1 (s,1H), 8.10 (d, J=8.2, 2H), 7.97 (d, J=8.2, 2H), 3.00 (t, J=7.3, 2H),1.70-1.8 (m, 2H), 1.22-1.42 (m, 10H), 0.88 (t, J=7.0, 3H).

Aldehyde 24

Heptyl 4-(formyl)benzoate

The title compound was prepared through a condensation between1-heptanol and 4-formylbenzoic acid. ¹H NMR (500 MHz, CDCl₃): δ 10.10(s, 1H), 8.20 (d, J=8.2, 2H), 7.95 (d, J=8.2, 2H), 4.35 (t, J=6.8, 2H),1.75-1.85 (m, 2H), 1.40-1.50 (m, 2H), 1.25-1.40 (m, 6H), 0.89 (t, J=7.0,3H).

Aldehydes 25 and 26 were prepared using procedures analogous to thosedescribed in Aldehyde 16 substituting the appropriately substitutedalcohol for 2-hydroxymethyl-4-phenyl-5-trifluoromethyl-thiophene in StepB:

Aldehyde 25

4-[(Benzothien-2-yl)methoxy]benzaldehyde

¹H NMR (500 MHz) δ 5.34 (s, 2H), 7.04 (d, J=8.7, 2H), 7.18 (s, 1H),7.25-7.30 (m, 4H), 7.76 (d, J=8.7, 2H), 9.82 (s, 1H).

Aldehyde 26

4-[(2,3-Diphenyl-2H-pyrazol-5-yl)methoxy]benzaldehyde

¹H NMR (500 MHz) δ 5.21 (s, 2H), 6.55 (s, 1H), 7.10 (d, J=8.7, 2H),7.14-7.17 (m, 5H), 7.21-7.30 (m, 5H), 7.79 (d, J=8.7, 2H), 9.82 (s, 1H).

PREPARATION OF EXAMPLES Example 1(R/S)-1-(4-(Nonyl)phenyl)methyl-3-hydroxy-pyrrolidin-3-yl)phosphonicacid

Step A: (R/S)-1-tert-Butoxycarbonyl-3-hydroxypyrrolidine

A solution of 2.5 g (28.7 mmol) of (R/S)-3-hydroxypyrrolidine in 10 mLof CH₂Cl₂ at 0° C. was treated with 6.89 g (31.6 mmol) ofdi-tert-butyl-dicarbonate in 2 mL CH₂Cl₂ and 0.35 g (2.8 mmol) of4-(N,N-dimethylamino) pyridine. After stirring for 10 min, the reactionwas warmed to rt and stirred overnight. The reaction was diluted with100 mL of CH₂Cl₂ and washed with 100 mL of 1N HCl and 100 mL of 1NNaHCO₃. The organic layer was dried over Na₂SO₄ and concentrated. Theresidue was purified on a 40M Biotage column using 7:3 v/vhexane/acetone as the eluant to afford 5.3 g (99%) of the titlecompound: R_(F): 0.26 (7:3 v/v hexane/acetone); ¹H-NMR (500 MHz) δ 1.45(s, 9H), 1.88-2.00 (m, 2H), 2.52 (br s, 1H), 3.29-3.50 (m, 4H), 4.42 (m,1H).

Step B: 1-tert-Butoxycarbonyl-3-oxo-pyrrolidine

A solution of 2.3 mL (26 mmol) of oxalyl chloride in 80 mL of CH₂Cl₂ at−78° C. was treated with 3.8 mL (53 mmol) of DMSO in 5 mL of CH₂Cl₂. Theresulting mixture was stirred cold for 5 min. A solution of 2.0 g (10.7mmol) of (R/S)-1-tert-butoxycarbonyl-3-hydroxypyrrolidine (from Step A)in 10 mL of CH₂Cl₂ was added. The resulting mixture was stirred for 30min, treated with 18.7 mL (107 mmol) of DIEA and warmed to 0° C. Afterstirring for 45 min, the reaction was quenched with H₂O and poured into100 mL of 1N HCl. After separating the layers, the organic layer waswashed with 100 mL sat'd NaCl, dried over Na₂SO₄ and concentrated. Theresidue was purified on a 40M Biotage column using 4:1 v/vhexane/acetone as the eluant to afford 1.9 g (96%) of the titlecompound: R_(F): 0.49 (7:3 v/v hexane/acetone); ¹H-NMR (500 MHz) δ 1.48(s, 9H), 2.58 (t, J=7.9, 2H), 3.71-3.78 (m, 4H).

Step C: (R/S)-1-tert-Butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl phosphonicacid, diethyl ester

A mixture of 1.9 g (10.3 mmol) of 1-tert-butoxycarbonyl-3-oxopyrrolidine(from Step B), 1.3 mL (10.3 mmol) of diethyl phosphite and 1.4 mL (10.3mmol) of TEA was stirred at 100° C. for 1.5 h. Volatiles were removedunder reduced pressure. The residue was purified on a 40M Biotage columnusing 13:7 v/v hexane/acetone as the eluant to afford 1.78 g (53%) ofthe title compound as a yellow oil: R_(F): 0.16 (7:3 v/vhexane/acetone); ¹H-NMR (500 MHz) δ 1.33 (t, J=7.0, 6H), 1.45 (s, 9H),2.08 (m, 1H), 2.18 (m, 1H), 3.47-3.64 (m, 4H), 4.13-4.22 (m, 4H).

Step D: (R/S)-3-Hydroxy-pyrrolidin-3-yl phosphonic acid, diethyl ester

A solution of 1.78 g (5.5 mmol) of(R/S)-1-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl phosphonic acid,diethyl ester (from Step C) in 2N HCl in EtOH was stirred at rt for 5.5h. The reaction was concentrated from CH₂Cl₂ several times. The crudeproduct was partitioned between aqueous NH₄OH and CHCl₃/isopropanol (3:1v/v). After separating phases, the aqueous layer was extracted with 3×CHCl₃/isopropanol (3:1 v/v). The combined organics were dried overNa₂SO₄ and concentrated. The residue was purified on a 40S Biotagecolumn using 90:10:1 v/v/v CH₂Cl₂/MeOH/NH₄OH as the eluant to afford thetitle compound as a light brown oil: ¹H-NMR (500 MHz) δ 1.35 (t, J=7.0,6H), 1.92 (m, 1H), 2.20 (m, 1H), 2.78-2.99 (m, 3H), 3.06 (dd, J=12.7,3.7, 1H), 3.13 (dd, J=12.7, 6.2, 1H), 3.20 (m, 1H), 4.16-4.23 (m, 4H).

Step E: (R/S)-1-(4-(Nonylphenyl)methyl-3-hydroxy-pyrrolidin-3-ylphosphonic acid, diethyl ester

A solution of 60 mg (0.23 mmol) of(R/S)-3-hydroxy-pyrrolidin-3-ylphosphonic acid, diethyl ester (from StepD) and 54 mg (0.23 mmol) of Aldehyde 1 in 1.5 mL of CH₂Cl₂ was treatedwith 73 mg (0.34 mmol) of sodium triacetoxyborohydride. After 3 h at rt,the reaction was diluted with 25 mL of CH₂Cl₂ and washed with 25 mL of1N NaHCO₃. After separating phases, the aqueous layer was extracted with25 mL of CH₂Cl₂. The combined organic layers were washed with 50 mL ofsat'd NaCl, dried over Na₂SO₄ and concentrated. The residue was purifiedby flash chromatography using 3:1 v/v hexane/acetone as the eluant toafford 33 mg (32%) of the title compound: R_(F): 0.31 (7:3 v/vhexane/acetone); ¹H-NMR (500 MHz) δ 0.89 (t, J=7.0, 3H), 1.27-1.36 (m,18H), 1.57-1.63 (m, 2H), 1.97 (m, 1H), 2.41-2.54 (m, 2H), 2.59 (t,J=7.7, 2H), 2.85-2.92 (m, 2H), 3.01 (m, 1H), 3.67 (ABq, J=13.1, 2H),4.16-4.23 (m, 4H), 7.12 (d, J=7.8, 2H), 7.24 (d, J=7.8, 2H).

Step F: (R/S)-1-(4-Nonylbenzyl)-3-hydroxypyrrolidin-3-ylphosphonic acid

A solution of 33 mg (0.075 mmol) of(R/S)-1-(4-nonylbenzyl)-3-hydroxypyrrolidin-3-ylphosphonic acid, diethylester (from Step E) in 1 mL of chloroform was treated with 0.053 mL(0.37 mmol) of iodotrimethylsilane. The reaction was allowed to stir atrt for 1 h. The reaction was quenched with MeOH and concentrated severaltimes from MeOH. The residue was purified using LC-2 to afford 4.6 mg(16%) of the title compound: ESI-MS 385 (M+H); LC-1: 3.01 min.

Examples 2-10

EXAMPLES 2-10 were prepared using procedures analogous to thosedescribed in EXAMPLE 1 substituting the appropriate Aldehyde in Step E.TMS-Br was substituted in Step F with substrates containing TMS-Isensitive functionality (See EXAMPLE 11, Step D). In EXAMPLES 5 and 6enantiomers were resolved after Step E by preparative chiral HPLC(Chiralpak AD 2×25 cm HPLC column, 9:1 v/v hexane/EtOH, flow rate=9.0mL/min, λ=210 nM).

EXAMPLE HPLC HPLC RT ESI-MS # R Method (min) (M + H) 2

LC-1 2.7 386 3

LC-1 2.7 386 4

LC-1 3.0 496 5Enantiomer 1

LC-1 2.8 430 ¹H-NMR (500 MHz, CD₃OD) δ 0.92(t, J=7.0, 3H), 1.20-1.54(m,9H), 1.79-1.84 (m, 2H), 2.23(m, 1H), 2.35(m, 1H), 2.43(m, 1H), 2.68(m,1H), 3.41-3.50(m, 2H), 3.58(m, 1H), 3.68(m, 1H), 3.75-3.79(m, 2H),4.04(t, J=6.4, 2H), 4.11-4.15(m, 2H), 4.38(ABq, J=12.9, 2H),7.02-7.09(m, 2H), 7.17(s, 1H) 6Enantiomer 2

LC-1 2.8 430 7

LC-1 3.1 544 ¹H-NMR (500 MHz, CD₃OD) δ 0.93(t, J=6.8, 3H), 1.20-1.46(m,9H), 1.55-1.61 (m, 2H), 1.86-1.92(m, 2H), 2.23-2.35(m, 2H), 2.72(m, 1H),3.47-3.79(br m, 3H), 4.06(t, J=6.4, 2H), 4.44-4.50(m, 2H), 7.86(s, 2H) 8

LC-1 2.6 398 9

LC-1 2.5 400 10 

LC-1 2.4 406

Example 11 (R/S)-1-(4-Nonylphenyl)methyl-pyrrolidin-3-yl phosphonic acid

Step A: (R/S)-1-Benzyl-pyrrolidin-3-yl phosphonic acid, diethyl ester

A solution of 6.0 g (36.6 mmol) of diethyl vinylphosphonate and 11 mL(44 mmol) of N-(methoxymethyl)-N-(trimethylsilylmethyl)benzylamine in150 mL of CH₂Cl₂ at 0° C. was stirred for 30 min. The reaction mixturewas washed with 150 mL of 1N NaHCO₃ and 150 mL of sat'd NaCl. Theorganic layer was dried over Na₂SO₄ and concentrated. The residue waspurified on a 40L Biotage column using 3:2 and 1:1 v/v hexane/acetone asthe gradient to afford 9.44 g (87%) of the title compound as a paleyellow oil: R_(F): 0.24 (3:2 v/v hexane/acetone); ¹H-NMR (500 MHz) δ1.32 (t, J=7.0, 6H), 2.04-2.12 (m, 2H), 2.39-2.58 (m, 3H), 2.83 (m, 1H),2.97 (m, 1H), 3.64 (s, 2H), 4.06-4.16 (m, 4H), 7.24-7.34 (m, 5H); ESI-MS298 (M+H); LC-1: 1.2 min.

Step B: (R/S)-Pyrrolidin-3-ylphosphonic acid, diethyl ester

A mixture of 3 g (10 mmol) of (R/S)-1-benzyl-pyrrolidin-3-ylphosphonicacid, diethyl ester (from Step A), 9.5 g (150 mmol) of ammonium formateand 1.0 g of 10% palladium on charcoal in 60 mL of MeOH was warmed to40° C. for 1.5 h. The reaction was cooled, filtered through a pad ofcelite and concentrated. The mixture was partitioned between 75 mL of 1NNaOH and 100 mL of CH₂Cl₂. After separating layers, the aqueous phasewas extracted with 3×100 mL of CH₂Cl₂. The combined organic layers weredried over Na₂SO₄ and concentrated. The residue was purified on a 40MBiotage column using 90:10:1 v/v/v CH₂Cl₂/MeOH/NH₄OH as the eluant toafford the title compound as a pale yellow oil: R_(F): 0.13 (95:5:0.5v/v/v CH₂Cl₂MeOH/NH₄OH); ¹H-NMR (500 MHz) δ 1.22 (t, J=7.1, 6H), 1.81(m, 1H), 1.95 (m, 1H), 2.25 (m, 1H), 2.73 (m, 1H), 2.89-2.99 (m, 3H),4.06-4.16 (m, 4H).

Step C: (R/S)-1-(4-Nonylphenyl)methyl-pyrrolidin-3-ylphosphonic acid,diethyl ester

A solution of 41 mg (0.19 mmol) of (R/S)-pyrrolidin-3-yl phosphonicacid, diethyl ester (from Step B) and 43 mg (0.18 mmol) of Aldehyde 1 in1 mL of CH₂Cl₂ was treated with 57 mg (0.27 mmol) of sodiumtriacetoxyborohydride. After stirring at rt overnight, the reaction wasdiluted with 25 mL of CH₂Cl₂ and washed with 25 mL of 1N NaHCO₃. Afterseparating phases, the aqueous layer was extracted with 25 mL of CH₂Cl₂.The combined organic layers were washed with 50 mL of sat'd NaCl, driedover Na₂SO₄ and concentrated. The residue was purified by flashchromatography using 49:1 v/v CH₂Cl₂/MeOH as the eluant to afford 67 mg(99%) of the title compound: R_(F): 0.39 (19:1 v/v CH₂Cl₂/MeOH); ¹H-NMR(500 MHz) δ 0.90 (t, J=7.0, 3H), 1.20-1.35 (m, 17H), 1.59-1.65 (m, 2H),2.04-2.13 (m, 3H), 2.41-2.62 (m, 5H), 2.85 (m, 1H), 2.99 (m, 1H), 3.62(s, 2H), 4.08-4.17 (m, 4H), 7.14 (d, J=8.0, 2H), 7.24 (d, J=8.0, 2H).

Step D: (R/S)-1-(4-Nonylbenzyl)-pyrrolidin-3-ylphosphonic acid

A solution of 67 mg (0.16 mmol) of(R/S)-1-(4-nonylbenzyl)-pyrrolidin-3-ylphosphonic acid, diethyl ester(from Step C) in 1 mL of acetonitrile was treated with 0.094 mL (0.71mmol) of bromotrimethylsilane. The reaction was allowed to stir at 80°C. for 1 h. The reaction was quenched with MeOH and concentrated severaltimes from MeOH. The residue was purified by LC-2 to afford 27 mg (46%)of the title compound: ESI-MS 368 (M+H); LC-1: 3.1 min.

Examples 12-17

EXAMPLES 12-17 were prepared using procedures analogous to thosedescribed in EXAMPLE 11 substituting the appropriate Aldehyde in Step C.In EXAMPLES 15 and 16 enantiomers were were resolved after Step E bypreparative chiral HPLC (Chiralcel OD 2×25 cm HPLC column, 19:1 v/vhexane/iPrOH, flow rate=9.0 mL/min, λ=210 nM).

EXAMPLE HPLC HPLC RT ESI-MS # R Method (min) (M + H) 12

LC-1 2.8 370 13

LC-1 2.7 370 14

— — — ¹H-NMR (500 MHz, CD₃OD) δ 0.92(t, J=7.0, 3H), 1.34-1.54(m, 10H),1.79-1.84 (m, 2H), 2.18(m, 1H), 2.32-2.45(m, 2H), 2.69(m, 1H), 2.88(m,1H), 3.22-3.37(m, 2H), 3.47-3.62(m, 2H), 3.73(m, 1H), 4.04(t, J=6.4,2H), 4.13(q, J=7.0, 2H), 4.32- 4.37(m, 2H), 7.02-7.08(m, 2H), 7.16(s,1H) 15Enantiomer 1

LC-1 3.2 528 ¹H-NMR (500 MHz, CD₃OD) δ 0.93(t, J=6.9, 3H), 1.34-1.46(m,8H), 1.55-1.61 (m, 2H), 1.86-1.95(m, 2H), 2.25-2.47(m, 2H), 2.72(m, 1H),3.28(m, 1H), 3.63-3.79 (m, 3H), 4.06(t, J=6.4, 2H), 4.44(s, 2H), 7.87(s,2H) 16Enantiomer 2

LC-1 3.1 528 17

LC-1 2.4 390

Example 18(R/S)-1-{4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzyl}-pyrrolidin-3-ylcarboxylic acid

Step A: (R/S)-1-Benzyl-pyrrolidin-3-yl carboxylic acid, benzyl ester

A solution of 10.0 g (61.6 mmol) of benzyl acrylate and 19 mL (74.2mmol) of N-(methoxymethyl)-N-(trimethylsilylmethyl)benzylamine in 75 mLof CH₂Cl₂ at 0° C. was treated with 0.5 mL (6.5 mmol) of TFA whilemaintaining the internal temperature at less than 3° C. The reaction waswarmed to rt and stirred for 2.5 h. The reaction mixture was washed with250 mL of 1N NaHCO₃ and 250 mL of sat'd NaCl. The organic layer wasdried over Na₂SO₄ and concentrated. The residue was purified on a 40LBiotage column using 19:1 v/v hexane/acetone as the eluant to afford 18g (99%) of the title compound as a light yellow oil: R_(F): 0.28 (9:1v/v hexane/acetone); ¹H-NMR (500 MHz) δ 2.15-2.20 (m, 2H), 2.60 (m, 1H),2.73-2.77 (m, 2H), 3.02 (m, 1H), 3.13 (m, 1H), 3.66-3.73 (m, 2H), 5.17(s, 2H), 7.28-7.42 (m, 5H).

Step B: (R/S)-1-Benzyloxycarbonyl-pyrrolidin-3-yl carboxylic acid,benzyl ester

A solution of 18 g (61 mmol) of (R/S)-1-benzyl-pyrrolidin-3-ylcarboxylic acid, benzyl ester (from Step A) in 100 mL of CH₂Cl₂ at 0° C.was treated with 21.3 mL (231 mmol) of benzyl chloroformate whilemaintaining the internal temperature at less than 6° C. The reaction wasallowed to warm to rt overnight. After 24 hours at rt, an additional 10mL (10.8 mmol) of benzyl chloroformate was added. After 24 hours ofstirring at rt, the reaction was concentrated. The residue was purifiedon a 40L Biotage column using 19:1 v/v hexane/acetone as the eluant toafford 8.42 g (39%) of the title compound as a colorless oil: R_(F):0.14 (9:1 v/v hexane/acetone); ¹H-NMR (500 MHz) δ 2.19-2.22 (m, 2H),3.15 (m, 1H), 3.45-3.75 (m, 4H), 5.13-5.20 (m, 4H), 7.33-7.41 (m, 10H).

Step C: (R/S)-Pyrrolidin-3-yl carboxylic acid

A mixture of 8.4 g (24.7 mmol) of(R/S)-1-benzyloxycarbonyl-pyrrolidin-3-yl carboxylic acid, benzyl ester(from Step B) and 2.86 g of 10% palladium on charcoal in 80 mL of MeOHwas hydrogenated at atmospheric pressure using a balloon of hydrogen for6.5 h. The reaction was filtered through a pad of Celite andconcentrated to afford 2.72 g (95%) of the title compound as a whitesolid: ¹H-NMR (500 MHz, CD₃OD) δ 2.17-2.26 (m, 2H), 3.03 (m, 1H),3.24-3.38 (m, 3H), 3.51 (m, 1H).

Step D:(R/S)-1-{4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzyl}-pyrrolidin-3-ylcarboxylic acid

A mixture of 17.5 mg (0.15 mmol) of (R/S)-pyrrolidin-3-yl carboxylicacid (from Step C), 78 mg (0.21 mmol) of Aldehyde 16 and 9 mg (0.14mmol) of sodium cyanoborohydride in 2 mL of MeOH was stirred at rtovernight. The reaction was concentrated and purified by flashchromatography using 19:1 v/v CH₂Cl₂/MeOH, then 85:15:1.5 v/v/vCH₂Cl₂/MeOH/NH₄OH as the eluant to afford 42 mg (63%) of the titlecompound as a white foam: R_(F): 0.29 (85:15:1.5 v/v/vCH₂Cl₂/MeOH/NH₄OH); ¹H-NMR (500 MHz, CD3OD) δ 2.23-2.35 (m, 2H), 3.09(m, 1H), 3.26-3.41 (m, 3H), 3.53 (m, 1H), 4.30 (ABq, J=13.0, 2H), 5.38(s, 2H), 7.13 (d, J=8.5, 2H), 7.22 (s, 1H), 7.39-7.45 (m, 5H), 7.48 (d,J=8.5, 2H); ESI-MS 462 (M+H); LC-1: 2.7 min.

Examples 19-33

EXAMPLES 19-33 were prepared using procedures analogous to thosedescribed in EXAMPLE 18 substituting the appropriate Aldehyde in Step D.

EXAMPLE HPLC HPLC RT ESI-MS # R Method (min) (M + H) 19

LC-1 2.8 332 ¹H-NMR (500 MHz) δ 0.91(t, J=6.9, 3H), 1.30-1.34(m, 12H),1.60-1.63(m, 2H), 2.33-2.41(m, 2H), 2.60-2.63(m, 2H), 3.09-3.29(m, 4H),3.73(m, 1H), 4.20(ABq, J= 12.5, 2H), 7.21(d, J=7.7, 2H), 7.44(d, J=7.7,2H) 20

LC-1 3.0 346 21

LC-1 3.0 334 ¹H-NMR (500 MHz, CD₃OD) δ 0.91(t, J=7.0, 3H), 1.31-1.50(m,10H), 1.75-1.80 (m, 2H), 2.22-2.33(m, 2H), 3.08(m, 1H), 3.25-3.40(m,3H), 3.52(m, 1H), 3.99(t, J= 6.4, 2H), 4.28(ABq, J=13.0, 2H), 6.97(d,J=8.6, 2H), 7.41(d, J=8.6, 2H) 22

LC-1 2.9 364 ¹H-NMR (500 MHz, CD₃OD) δ 0.91(t, J=6.9, 3H), 1.31-1.51(m,10H), 1.76-1.82 (m, 2H), 2.24-2.37(m, 2H), 3.17(m, 1H), 3.29-3.43(m,3H), 3.56(m, 1H), 3.87(s, 3H), 4.01(t, J=6.5, 2H), 4.29(ABq, J=12.8,2H), 6.98(d, J=8.2, 1H), 7.03(dd, J= 8.2, 1.7, 1H), 7.12(d, J=1.7, 1H)23

LC-1 3.3 348 24

LC-1 3.5 384 25

LC-1 3.2 368 26

LC-1 3.2 368 27

LC-1 2.9 358 28

LC-1 3.2 500 ¹H-NMR (500 MHz, CD₃OD) δ 2.26-2.37(m, 2H), 3.13(m, 1H),3.25-3.43(m, 3H), 3.52(m, 1H), 4.37(ABq, J=12.9, 2H), 7.49-7.50(m, 5H),7.69(d, J=8.1, 2H), 8.00 (s, 1H), 8.16(d, J=8.1, 2H) 29

LC-1 3.0 362 EXAMPLE 29 was prepared by catalytic hydrogenation ofEXAMPLE 27 using a procedure analogous to that described in EXAMPLE 18,Step C. 30

LC-1 2.9 448 ¹H-NMR (500 MHz, CD₃OD) δ 2.23-2.34(m, 2H), 3.09(m, 1H),3.25-3.40(m, 3H), 3.53(m, 1H), 4.30(ABq, J=13.0, 2H), 5.31(s, 2H),7.14(d, J=8.6, 2H), 7.48(d, J= 8.6, 2H), 7.94(s, 1H), 8.07(s, 2H) 31

— — 368 32

— — 352 33

— — 454

Example 35 (R/S)-1-(4-Nonylphenyl)methyl-3-fluoro-pyrrolidin-3-ylcarboxylic acid

Step A: (R/S)-1-Benzyl-pyrrolidin-3yl carboxylic acid, methyl ester

The title compound was prepared using a procedure analogous to thatdescribed in EXAMPLE 18, Step A substituting methyl acrylate for benzylacrylate: R_(F): 0.29 (9:1 v/v hexane/acetone); ¹H-NMR (500 MHz) δ2.10-2.14 (m, 2H), 2.55 (m, 1H), 2.66 (m, 1H), 2.75 (m, 1H), 2.94 (m,1H), 3.06 (m, 1H), 3.65 (s, 2H), 3.69 (s, 3H), 7.25-7.35 (m, 5H).

Step B: (R/S)-Pyrrolidin-3-yl carboxylic acid, methyl esterhydrochloride salt

A solution of 0.52 g (2.3 mmol) of (R/S)-1-benzyl-pyrrolidin-3-ylcarboxylic acid, methyl ester (from Step A) in 5 mL of1,2-dichloroethane was treated with 0.3 mL (2.7 mmol) of 1-chloroethylchloroformate (ACE-Cl). The resulting mixture was stirred at rt for 3 h,then at reflux for 30 min. The reaction was cooled and concentrated. Theresidue was warmed to reflux in 5 mL of MeOH for 1 h. The reaction wascooled and concentrated. The crude product was used in Step C withoutfurther purification.

Step C: (R/S)-1-(4-Nonylphenyl)methyl-pyrrolidin-3-yl carboxylic acid,methyl ester

The title compound was prepared using an analogous procedure describedin EXAMPLE 1, Step E substituting (R/S)-pyrrolidin-3-yl carboxylic acid,methyl ester hydrochloride salt (from Step B) for(R/S)-3-hydroxypyrrolidin-3-ylphosphonic acid, diethyl ester and usingDIEA to neutralize the hydrochloride salt: R_(F): 0.44 (4:1 v/vhexane/acetone); ¹H-NMR (500 MHz) δ 0.91 (t, J=6.9, 3H), 1.30-1.35 (m,12H), 1.60-1.66 (m, 2H), 2.13-2.17 (m, 2H), 2.54-2.69 (m, 4H), 2.80 (m,1H), 2.99 (m, 1H), 3.09 (m, 1H), 3.66 (s, 2H), 3.72 (s, 3H), 7.16 (d,J=8.0, 2H), 7.27 (d, J=8.0, 2H).

Step D: (R/S)-1-(4-Nonylphenyl)methyl-3-fluoropyrrolidin-3-yl carboxylicacid, methyl ester

To a solution of 1 mL (0.32 mmol) of 0.32M lithium diisopropylamide inTHF at −78° C. was added 90 mg (0.26 mmol) of (R/S)-1-1-(4-nonylphenyl)methylbenzyl)-pyrrolidin-3-yl carboxylic acid, methyl ester (from StepC) in 1.5 mL of THF while maintaining the internal temperature at less−70° C. After 15 min, 111 mg (0.35 mmol) of fluorobenzenesulfonimide in0.5 mL THF was added while maintaining the internal temperature at less−68° C. After stirring for 15 min, the reaction was warmed to 0° C. andquenched with 0.1N HCl. The reaction mixture was poured into 50 mL ofEt₂O and washed with 50 mL of 1N NaHCO₃ and 50 mL of sat'd NaCl. Theorganic phase was dried over MgSO₄ and concentrated. The residue waspurified by flash chromatography using 19:1 v/v hexane/acetone as theeluant to afford 47 mg (50%) of the title compound as a colorless film:R_(F): 0.36 (9:1 v/v hexane/acetone); ¹H-NMR (500 MHz) δ 0.91 (t, J=6.8,3H), 1.30-1.35 (m, 12H), 1.60-1.66 (m, 2H), 2.28 (m, 1H), 2.49 (m, 1H),2.62 (t, J=7.8, 2H), 2.69 (m, 1H), 2.95-3.10 (m, 3H), 3.69 (ABq, J=12.8,2H), 3.83 (s, 3H), 7.16 (d, J=7.8, 2H), 7.27 (d, J=7.8, 2H).

Step E: (R/S)-1-(4-Nonylphenyl)methyl-3-fluoropyrrolidin-3-yl carboxylicacid

A solution of 46 mg (0.12 mmol) of(R/S)-1-(4-nonylphenyl)methyl-3-fluoropyrrolidin-3-yl carboxylic acid,methyl ester (from Step D) in 3 mL of EtOH was treated with 0.16 mL(0.16 mmol) of 1N NaOH and stirred overnight at rt. The reaction wasneutralized with 2 mL of pH 7 buffer and concentrated. Toluene was addedand the resulting mixture was concentrated. The residue was purified byflash chromatography using 19:1 v/v CH₂Cl₂/MeOH, then 90:10:1 v/v/vCH₂Cl₂/MeOH/NH₄OH as the eluant to afford 38 mg (86%) of the titlecompound as a white, waxy solid: R_(F): 0.21 (85:15:1.5 v/v/vCH₂Cl₂/MeOH/NH₄OH); ¹H-NMR (500 MHz) δ 0.79 (t, J=6.8, 3H), 1.18-1.23(m, 12H), 1.48-1.52 (m, 2H), 2.30 (m, 1H), 2.47-2.59 (m, 3H), 3.29-3.44(m, 3H), 3.73 (m, 1H), 3.87 (br m, 1H), 4.17 (ABq, J=12.9, 2H), 7.12 (d,J=7.9, 2H), 7.28 (d, J=7.9, 2H); ESI-MS 350 (M+H); LC-1: 3.3 min.

Example 36 (R/S)-1-(4-Nonylphenyl)methyl-3-hydroxypyrrolidin-3-ylcarboxylic acid

Step A: (R/S) 1-(4-Nonylphenyl)methyl-3-hydroxypyrrolidin-3-ylcarboxylic acid methyl ester

To a solution of 0.52 mL (0.52 mmol) of 1.0M sodium hexamethylsilazidein THF at −78° C. was added 153 mg (0.44 mmol) of(R/S)-1-(4-nonylphenyl)methyl-pyrrolidin-3-yl carboxylic acid, methylester (from EXAMPLE 34, Step C) in 1 mL of THF while maintaining theinternal temperature at less −72° C. After 20 min, 172 mg (0.65 mmol) of2-(phenylsulfonyl)-3-phenyloxaziridine (Davis Reagent) in 1 mL of THFwas added while maintaining the internal temperature at less −69° C.After stirring for 1.25 h at −78° C., the reaction was quenched with 1NNaHCO₃ and warmed to rt. After removing volatiles under reducedpressure, the reaction mixture was diluted with 50 mL of 1N NaHCO₃ and50 mL of sat'd NaCl. The aqueous phase was extracted with 3×50 mL ofCH₂Cl₂. The combined organic layers were dried over Na₂SO₄ andconcentrated. The residue was purified by flash chromatography using 4:1v/v hexane/EtOAc and 4:1 v/v hexane/acetone as the gradient to afford 11mg (7%) of the title compound as a colorless film: R_(F): 0.39 (4:1 v/vhexane/acetone); ¹H-NMR (500 MHz) δ 0.90 (t, J=6.8, 3H), 1.28-1.33 (m,12H), 1.59-1.64 (m, 2H), 2.02 (m, 1H), 2.42 (m, 1H), 2.60 (t, J=7.8,2H), 2.67 (m, 1H), 2.86 (ABq, J=10.1, 2H), 2.97 (m, 1H), 3.69 (s, 2H),3.82 (s, 3H), 7.14 (d, J=7.9, 2H), 7.26 (d, J=7.9, 2H).

Step B: (R/S)-1-(4-Nonylphenyl)methyl-3-hydroxypyrrolidin-3-ylcarboxylic acid

The title compound was prepared using an analogous procedure describedin EXAMPLE 34, Step E substituting(R/S)-1-(4-nonylphenyl)methyl-3-hydroxypyrrolidin-3-yl carboxylic acid,methyl ester (from Step A) for(R/S)-1-(4-nonylphenyl)methyl-3-fluoropyrrolidin-3-yl carboxylic acid,methyl ester: R_(F): 0.15 (90:10:1 v/v/v CH₂Cl₂/MeOH/NH₄OH); ¹H-NMR (500MHz, CD₃OD) δ 0.89 (t, J=6.9, 3H), 1.28-1.33 (m, 12H), 1.60-1.63 (m,2H), 2.10 (m, 1H), 2.49 (m, 1H), 2.64 (t, J=7.7, 2H), 3.25 (m, 1H),3.49-3.62 (m, 3H), 4.38 (ABq, J=13.0, 2H), 7.28 (d, J=7.8, 2H), 7.42 (d,J=7.8, 2H); ESI-MS 348 (M+H); LC-1: 3.0 min.

Example 37 (R/S)-1-(4-Nonylphenyl)methyl-pyrrolidin-3-yl acetic acid

Step A: (R/S)-1-(4-Nonylphenyl)methyl-pyrrolidin-3-ylacetic acid,tert-butyl ester

The title compound was prepared using an analogous procedure describedin EXAMPLE 1, Step E substituting (R/S)-pyrrolidin-3-yl acetic acid,tert-butyl ester hydrochloride salt for(R/S)-3-hydroxypyrrolidin-3-ylphosphonic acid, diethyl ester and usingDIEA to neutralize the hydrochloride salt: R_(F): 0.53 (4:1 v/vhexane/acetone); ¹H-NMR (500 MHz) δ 0.90 (t, J=6.8, 3H), 1.28-1.64 (m,25H), 2.09 (m, 1H), 2.26-2.37 (m, 3H), 2.58-2.69 (m, 4H), 2.89 (m, 1H),3.61-3.64 (m, 2H), 7.14 (d, J=7.4, 2H), 7.26 (d, J=7.4, 2H).

Step B: (R/S)-1-(4-Nonylphenyl)methyl-pyrrolidin-3-yl acetic acid

A solution of 50.5 mg (0.12 mmol) of(R/S)-1-(4-nonylphenyl)methyl-pyrrolidin-3-yl acetic acid, tert-butylester (from Step A) in formic acid at 55° C. was stirred for 2.25 h.Volatiles were removed under reduced pressure. The residue was purifiedby flash chromatography using 19:1 v/v CH₂Cl₂/MeOH, then 85:15:1.5 v/v/vCH₂Cl₂/MeOH/NH₄OH as the eluant to afford 41 mg (94%) of the titlecompound as a sticky, waxy film: R_(F): 0.31 (85:15:1.5 v/v/vCH₂Cl₂/MeOH/NH₄OH); ¹H-NMR (500 MHz, CD₃OD) δ 0.90 (t, J=6.9, 3H),1.29-1.33 (m, 12H), 1.61-1.64 (m, 2H), 1.77 (m, 1H), 2.26-2.45 (m, 3H),2.64 (t, J=7.7, 2H), 2.71 (m, 1H), 3.08 (m, 1H), 3.23 (m, 1H), 3.38-3.44(m, 2H), 4.28 (s, 2H), 7.28 (d, J=8.1, 2H), 7.39 (d, J=8.1, 2H); ESI-MS346 (M+H); LC-1: 3.3 min.

Example 38(R/S)-1-{4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzyl}-pyrrolidin-3-ylaceticacid

The title compound was prepared using procedures analogous to thosedescribed in EXAMPLE 36 substituting Aldehyde 16 for Aldehyde 1 in StepA: R_(F): 0.29 (85:15:1.5 v/v/v CH₂Cl₂/MeOH/NH₄OH); ¹H-NMR (500 MHz,CD₃OD) δ 1.77 (m, 1H), 2.26-2.46 (m, 3H), 2.71 (m, 1H), 3.07 (m, 1H),3.23 (m, 1H), 3.37-3.34 (m, 2H), 4.28 (s, 2H), 5.38 (s, 2H), 7.13 (d,J=8.7, 2H), 7.23 (s, 1H), 7.40-7.47 (m, 7H); ESI-MS 476 (M+H); LC-1: 3.0min.

Example 39 (R/S)-5-[1-(4-Nonylphenyl)methylpyrrolidin-3-yl]-1H-tetrazole

Step A: (R/S)-1-Benzyloxycarbonyl-3-cyano pyrrolidine

The title compound was prepared using analogous procedures described inEXAMPLE 18 (Steps A and B) substituting acrylonitrile for benzylacrylate in Step A: R_(F): 0.19 (4:1 v/v hexane/acetone); ¹H-NMR (500MHz) δ 2.18-2.28 (m, 2H), 3.12 (m, 1H), 3.53 (m, 1H), 3.61-3.78 (m, 3H),5.16 (d, J=3.0, 2H), 7.32-7.42 (m, 5H).

Step B: (R/S)-5-[1-Benzyloxycarbonyl-pyrrolidin-3-yl]-1H-tetrazole

A mixture of 1.8 g (7.8 mmol) of (R/S)-1-benzyloxycarbonyl-3-cyanopyrrolidine (from Step A), 1.5 g (23 mmol) of sodium azide and 1.25 g(23 mmol) of ammonium chloride in 70 mL of DMF was stirred at 105° C.overnight. After cooling to rt, the reaction was poured into 150 mL ofCH₂Cl₂ and washed with 150 mL of 1N HCl and 2×150 mL of H₂O. The organicphase was dried over MgSO₄ and concentrated. The residue was purified ona 40M Biotage column using 80:20:1 v/v/v CH₂Cl₂/EtOAc/HOAc as the eluantto afford 670 mg (31%) of the title compound: R_(F): 0.23 (80:20:1 v/v/vCH₂Cl₂/EtOAc/HOAc); ¹H-NMR (500 MHz) δ 2.29, 2.48 (2m, 2H), 3.54-4.03(m, 5H), 5.14-5.24 (m, 2H), 7.30-7.37 (m, 5H), 10.43 (br, 1H).

Step C: (R/S)-5-(Pyrrolidin-3-yl)-1H-tetrazole

A mixture of 662 mg (2.4 mmol) of(R/S)-5-[1-benzyloxycarbonyl-pyrrolidin-3-yl]-1H-tetrazole (from Step B)and 220 mg of 10% palladium on charcoal in 5 mL of MeOH was hydrogenatedat atmospheric pressure using a balloon of hydrogen for 3 h. Thereaction was filtered through a pad of Celite and concentrated to affordthe title compound as a white solid: ¹H-NMR (500 MHz, CD₃OD) δ 2.27 (m,1H), 2.49 (m, 1H), 3.39-3.51 (m, 3H), 3.70 (m, 1H), 3.85 (m, 1H).

Step D: (R/S)-5-[1-(4-Nonylbenzyl)methyl-pyrrolidin-3-yl]-1H-tetrazole

The title compound was prepared using an analogous procedure describedin EXAMPLE 18, Step D substituting(R/S)-5-(pyrrolidin-3-yl)-1H-tetrazole (from Step C) for(R/S)-pyrrolidin-3-yl carboxylic acid: 1H-NMR (500 MHz, CD₃OD) δ 0.89(t, J=7.0, 3H), 1.28-1.33 (m, 12H), 1.60-1.63 (m, 2H), 2.33 (m, 1H),2.55 (m, 1H), 2.64 (t, J=7.6, 2H), 3.47-3.55 (m, 3H), 3.76 (m, 1H), 3.92(m, 1H), 4.40 (s, 2H), 7.29 (d, J=8.0, 2H), 7.42 (d, J=8.0, 2H); ESI-MS356 (M+H); LC-1: 3.3 min.

Example 401-{4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzyl}-3-azetidinecarboxylicacid

The title compound was prepared by treating a mixture of 0.12 mmol of3-azetidinecarboxylic acid, 0.1 mmol of Aldehyde 16, 0.007 mL (0.12mmol) of acetic acid in 2 mL of MeOH with 10 mg (0.16 mmol) of sodiumcyanoborohydride and stirring the resulting mixture at rt for 3 h. Theproduct was purified using LC-2: ¹H NMR (500 MHz, CD₃OD) δ 3.34-3.37 (m,1H), 4.08 (app s, 2H), 4.10 (app s, 2H), 4.22 (s, 2H), 4.86 (s, 2H),5.35 (s, 2H), 7.10 (app d, J=8.0, 2H), 7.20 (s, 1H), 7.39-7.43 (5H).

Examples 41-45

EXAMPLES 41-45 were prepared using procedures analogous to thatdescribed in EXAMPLE 41 substituting the appropriate Aldehyde forAldehyde 16.

EXAMPLE HPLC HPLC RT ESI-MS # R Method (min) (M + H) 41

LC-1 3.3 318 ¹H-NMR (500 MHz, CD₃OD) δ 0.89(t, J=6.8, 3H), 1.28-1.32(m,12H), 1.60-1.62 (m, 2H), 2.63(t, J=7.7, 2H), 3.37(m, 1H), 4.12(s, 2H),4.13(s, 2H), 4.27(s, 2H), 7.27(d, J=8.0, 2H), 7.35(d, J=8.0, 2H) 42

LC-1 2.9 434 ¹H-NMR (500 MHz, CD₃OD) δ 3.35(m, 1H), 4.14(s, 2H), 4.16(s,2H), 4.28(s, 2H), 5.31(s, 2H), 7.14(d, J=8.6, 2H), 7.42(d, J=8.6, 2H),7.94(s, 1H), 8.07(s, 2H) 43

LC-1 2.4 405 44

— — 440 45

— — 338

Examples 46-53

The following compounds were prepared by treating a mixture of 0.12 mmolof either azetidine-3-carboxylic acid or (±)-pyrroldine-3-carboxylicacid, 0.1 mmol of Aldehyde, 7 μL (0.12 mmol) of acetic acid in 2 mL ofMeOH with 10 mg (0.16 mmol) of sodium cyanoborohydride and stirring theresulting mixture at rt for 1-3 h. The reaction mixtures were purifiedusing LC-2.

EXAMPLE Amino acid Aldehyde # LC-1 MS 46

19 2.9 min 496 (M + H) 47

19 2.9 min 482 (M + H) 48

18 3.1 min 496 (M + H) 49

18 3.1 min 482 (M + H) 50

21 2.9 min 492 (M + H) 51

21 2.9 min 478 (M + H) 52

20 3.1 min 476 (M + H) 53

20 3.1 min 462 (M + H) 54

15 3.2 min 485 (M + H)

Example 55 (3S,4R or3R,4S)-1-(4-Nonylbenzyl)4-trifluoromethylpyrrolidin-3-yl carboxylic acid

Step A: 4-(Nonyl)benzylamine

4-Nonylbenzoyl chloride (6 g, 20 mmol) and NH₄OAc (6 g,) were suspendedin acetone (100 mL) and stirred for 1 h at rt. Water (50 mL) was addedand the mixture filtered. The residue was washed with water and dried.The resulting crude amide (2.47 g,˜10 mmol) was dissolved in THF (5 mL)and borane dimethylsulfide complex (10 mL of 2M solution, 20 mmol) wasadded dropwise, while warming to reflux. The mixture was heated for 1 h.then cooled in an ice bath. Methanol (2.5 mL) was added dropwise,followed by 1N HCl in ether (11 mL). The white precipitate of the HClsalt of the benzyl amine was filtered off and washed with ether. The HClsalt was taken up in 2.5N NaOH and ether and the organic layer wasseparated and dried over Na₂SO₄. Evaporation afforded 1.3 g of the titlecompound.

Step B: N-(Methoxymethyl)-N-(trimethylsilylmethyl)-(4-nonyl)benzylamine

A solution of 1.3 g (6 mmol) of 4-(nonyl)benzylamine (from Step A) and700 mg (6 mmol) of chloromethyltrimethylsilane in 5 mL of DMSO wasstirred at 90° C. for 3 h, then at rt for 16 h. The mixture waspartitioned between MTBE and 1N NaOH. The organic layer was separated,washed with sat'd NaCl, dried and concentrated. Flash chromatographyusing 9:1 v/v hexane/EtOAc as the eluant afforded 700 mg ofN-(trimethylsilylmethyl)-4-(nonyl)benzylamine.

A mixture of the crude N-(trimethylsilylmethyl)-4-(nonyl)benzylamine,140 mg of paraformaldehyde and 15 mg of powdered NaOH in 5 mL of MeOHwas stirred at 40° C. for 1 h. The mixture was diluted with ether andaged for 16 h. The mixture was concentrated and dried to afford 700 mgof the title compound: ¹H NMR (500 MHz, CD₃OD) δ: 7.25 (m, 2H); 7.15 (m,2H); 4.03 (m, 2H); 3.74 (m, 2H); 3.28 (m, 2H); 2.61 (m, 2H); 2.22 (m,2H); 1.63 (m, 4H); 1.30 (m, 14H); 0.90 (m, 3H); 0.08 (m, 9H).

Step C:1-(4-(Nonyl)phenyl)methyl-3-(R/S)-carboxy-4-(R/S)-trifluoromethylpyrrolidine

A solution of 50 mg (0.14 mmol) ofN-(methoxymethyl)-N-(trimethylsilylmethyl)-(4-nonyl)benzylamine (fromStep B) and 20 mg (0.14 mmol) of trans-4,4,4-trifluoro-2-butenoic acid(0.137 mmol) in 1 mL of CH₂Cl₂ was treated with 1 drop of TFA and theresulting mixture was heated at 35° C. for 1 h. The reaction was cooled,concentrated then and then purified using LC-2 to afford the titlecompound: ¹H NMR (500 MHz, CD₃OD) δ 7.25 (d, J=8, 2H); 7.19 (d, J=8,2H); 3.87 (m, 2H); 3.54 (m, 1H); 3.27 (m, 4H); 2.93 (m, 1H); 2.61 (m,2H); 1.62 (m, 2H); 1.30 (m, 14H); 0.90 (t, J=6.7, 3H); ESI-MS 400.3(M+H).

Examples 56-59

EXAMPLES 56-58 were prepared using procedures analogous to thosedescribed in EXAMPLE 55 substituting the appropriate α,β-unsaturatedacid in Step C.

EXAMPLE ESI-MS # X Y (M + H) 56 H CF₃ 400.3 ¹H NMR (500 MHz, CD₃OD) δ:7.43(d, J=8 Hz, 2H); 7.29(d, J=8 Hz 2H); 4.35(s, 2H); 4.04(d, J=12 Hz,1H); 3.46(m, 1H); 2.65(m, 3H); 2.42(m, 1H); 1.62(m, 2H); 1.30(m, 14H);0.90(t, J=6.7 3H) 57 CO₂H H 375.3 ¹H NMR (500 MHz, CD₃OD) δ: 7.35(m, ,4H); 4.4(m, 1H); 4.12(m, 2H); 3.64(m, 1H); 2.69(m, 5H); 1.64(m, 1H);1.30(m, 14H); 0.90(m, 3H) 58 H CH₂CO₂H 390.3 ¹H NMR (500 MHz, CD₃OD) δ:7.36(m, , 4H); 4.43(m, , 1H); 4.14(m, 3H); 3.79(m, 1H); 3.50(m, 1H);3.09(m, 2H); 2.70(m, 8H); 3.18(m, 1H); 2.65(m, 2H); 2.3 (m, 2H); 1.61(m,2H); 1.29(M, 14H); 0.89(m, 3H)Biological Activity

The S1P₁/Edg1, S1P₃/Edg3, S1P₂/Edg5, S1P₄/Edg6 or S1P₅/Edg8 activity ofthe compounds of the present invention can be evaluated using thefollowing assays:

Ligand Binding to Edg/S1P Receptors Assay

³³P-sphingosine-1-phosphate was synthesized enzymatically from γ³³P-ATPand sphingosine using a crude yeast extract with sphingosine kinaseactivity in a reaction mix containing 50 mM KH₂PO₄, 1 mMmercaptoethanol, 1 mM Na₃VO₄, 25 mM KF, 2 mM semicarbazide, 1 mMNa₂EDTA, 5 mM MgCl₂, 50 mM sphingosine, 0.1% TritonX-114, and 1 mCiγ³³P-ATP (NEN; specific activity 3000 Ci/mmol). Reaction products wereextracted with butanol and ³³P-sphingosine-1-phosphate was purified byHPLC.

Cells expressing EDG/S1P receptors were harvested with enzyme-freedissociation solution (Specialty Media, Lavallette, N.J.). They werewashed once in cold PBS and suspended in binding assay buffer consistingof 50 mM HEPES-Na, pH 7.5, 5 mM MgCl₂, 1 mM CaCl₂, and 0.5% fattyacid-free BSA. ³³P-sphingosine-1-phosphate was sonicated with 0.1 nMsphingosine-1-phosphate in binding assay buffer; 100 μl of the ligandmixture was added to 100 μl cells (1×10⁶ cells/ml) in a 96 wellmicrotiter dish. Binding was performed for 60 min at room temperaturewith gentle mixing. Cells were then collected onto GF/B filter plateswith a Packard Filtermate Universal Harvester. After drying the filterplates for 30 min, 40 μl of Microscint 20 was added to each well andbinding was measured on a Wallac Microbeta Scintillation Counter.Non-specific binding was defined as the amount of radioactivityremaining in the presence of 0.5 μM cold sphingosine-1-phosphate.

Alternatively, ligand binding assays were performed on membranesprepared from cells expressing Edg/S1P receptors. Cells were harvestedwith enzyme-free dissociation solution and washed once in cold PBS.Cells were disrupted by homogenization in ice cold 20 mM HEPES pH 7.4,10 mM EDTA using a Kinematica polytron (setting 5, for 10 seconds).Homogenates were centrifuged at 48,000×g for 15 min at 4° C. and thepellet was suspended in 20 mM HEPES pH 7.4, 0.1 mM EDTA. Following asecond centrifugation, the final pellet was suspended in 20 mM HEPES pH7.4, 100 mM NaCl, 10 mM MgCl₂. Ligand binding assays were performed asdescribed above, using 0.5 to 2 μg of membrane protein.

Agonists and antagonists of Edg/S1P receptors can be identified in the³³P-sphingosine-1-phosphate binding assay. Compounds diluted in DMSO,methanol, or other solvent, were mixed with probe containing³³P-sphingosine-1-phosphate and binding assay buffer in microtiterdishes. Membranes prepared from cells expressing Edg/S1P receptors wereadded, and binding to ³³P-sphingosine-1-phosphate was performed asdescribed. Determination of the amount of binding in the presence ofvarying concentrations of compound and analysis of the data bynon-linear regression software such as MRLCalc (Merck ResearchLaboratories) or PRISM (GraphPad Software) was used to measure theaffinity of compounds for the receptor. Selectivity of compounds forEdg/S1P receptors was determined by measuring the level of³³P-sphingosine-1-phosphate binding in the presence of the compoundusing membranes prepared from cells transfected with each respectivereceptor (S1P₁/Edg1, S1P₃/Edg3, S1P₂/Edg5, S1P₄/Edg6, S1P₅/Edg8).

³⁵S-GTPγS Binding Assay

Functional coupling of S1P/Edg receptors to G proteins was measured in a³⁵S-GTPγS binding assay. Membranes prepared as described in the LigandBinding to Edg/S1P Receptors Assay (1-10 μg of membrane protein) wereincubated in a 200 μl volume containing 20 mM HEPES pH 7.4, 100 mM NaCl,10 mM MgCl₂, 5 μM GDP, 0.1% fatty acid-free BSA (Sigma, catalog A8806),various concentrations of sphingosine-1-phosphate, and 125 pM ³⁵S-GTPγS(NEN; specific activity 1250 Ci/mmol) in 96 well microtiter dishes.Binding was performed for 1 hour at room temperature with gentle mixing,and terminated by harvesting the membranes onto GF/B filter plates witha Packard Filtermate Universal Harvester. After drying the filter platesfor 30 min, 40 μl of Microscint 20 was added to each well and bindingwas measured on a Wallac Microbeta Scintillation Counter.

Agonists and antagonists of S1P/Edg receptors can be discriminated inthe ³⁵S-GTPγS binding assay. Compounds diluted in DMSO, methanol, orother solvent, were added to microtiter dishes to provide final assayconcentrations of 0.01 nM to 10 μM. Membranes prepared from cellsexpressing S1P/Edg receptors were added, and binding to ³⁵S-GTPγS wasperformed as described. When assayed in the absence of the naturalligand or other known agonist, compounds that stimulate ³⁵S-GTPγSbinding above the endogenous level were considered agonists, whilecompounds that inhibit the endogenous level of ³⁵S-GTPγS binding wereconsidered inverse agonists. Antagonists were detected in a ³⁵S-GTPγSbinding assay in the presence of a sub-maximal level of natural ligandor known S1P/Edg receptor agonist, where the compounds reduced the levelof ³⁵S-GTPγS binding. Determnination of the amount of binding in thepresence of varying concentrations of compound was used to measure thepotency of compounds as agonists, inverse agonists, or antagonists ofS1P/Edg receptors. To evaluate agonists, percent stimulation over basalwas calculated as binding in the presence of compound divided by bindingin the absence of ligand, multiplied by 100. Dose response curves wereplotted using a non-linear regression curve fitting program MRLCalc(Merck Research Laboratories), and EC₅₀ values were defined to be theconcentration of agonist required to give 50% of its own maximalstimulation. Selectivity of compounds for S1P/Edg receptors wasdetermined by measuring the level of ³⁵S-GTPγS binding in the presenceof compound using membranes prepared from cells transfected with eachrespective receptor.

Intracellular Calcium Flux Assay

Functional coupling of S1P/Edg receptors to G protein associatedintracellular calcium mobilization was measured using FLIPR(Fluorescence Imaging Plate Reader, Molecular Devices). Cells expressingS1P/Edg receptors were harvested and washed once with assay buffer(Hanks Buffered Saline Solution (BRL) containing 20 mM HEPES, 0.1% BSAand 710 μg/ml probenicid (Sigma)). Cells were labeled in the same buffercontaining 500 nM of the calcium sensitive dye Fluo-4 (Molecular Probes)for 1 hour at 37° C. and 5% CO₂. The cells were washed twice with bufferbefore plating 1.5×10⁵ per well (90 μl) in 96 well polylysine coatedblack microtiter dishes. A 96-well ligand plate was prepared by dilutingsphingosine-1-phosphate or other agonists into 200 μl of assay buffer togive a concentration that was 2-fold the final test concentration. Theligand plate and the cell plate were loaded into the FLIPR instrumentfor analysis. Plates were equilibrated to 37° C. The assay was initiatedby transferring an equal volume of ligand to the cell plate and thecalcium flux was recorded over a 3 min interval. Cellular response wasquantitated as area (sum) or maximal peak height (max). Agonists wereevaluated in the absence of natural ligand by dilution of compounds intothe appropriate solvent and transfer to the Fluo-4 labeled cells.Antagonists were evaluated by pretreating Fluo-4 labeled cells withvarying concentrations of compounds for 15 min prior to the initiationof calcium flux by addition of the natural ligand or other S1P/Edgreceptor agonist.

Preparation of Cells Expressing S1P/Edg Receptors

Any of a variety of procedures may be used to clone S1P₁/Edg1,S1P₃/Edg3, S1P₂/Edg5, S1P₄/Edg6 or S1P₅/Edg8. These methods include, butare not limited to, (1) a RACE PCR cloning technique (Frohman, et al.,1988, Proc. Natl. Acad. Sci. USA 85: 8998-9002). 5′ and/or 3′ RACE maybe performed to generate a full-length cDNA sequence; (2) directfunctional expression of the Edg/S1P cDNA following the construction ofan S1P/Edg-containing cDNA library in an appropriate expression vectorsystem; (3) screening an S1P/Edg-containing cDNA library constructed ina bacteriophage or plasmid shuttle vector with a labeled degenerateoligonucleotide probe designed from the amino acid sequence of theS1P/Edg protein; (4) screening an S1P/Edg-containing cDNA libraryconstructed in a bacteriophage or plasmid shuttle vector with a partialcDNA encoding the S1P/Edg protein. This partial cDNA is obtained by thespecific PCR amplification of S1P/Edg DNA fragments through the designof degenerate oligonucleotide primers from the amino acid sequence knownfor other proteins which are related to the S1P/Edg protein; (5)screening an S1P/Edg-containing cDNA library constructed in abacteriophage or plasmid shuttle vector with a partial cDNA oroligonucleotide with homology to a mammalian S1P/Edg protein. Thisstrategy may also involve using gene-specific oligonucleotide primersfor PCR amplification of S1P/Edg cDNA; or (6) designing 5′ and 3′ genespecific oligonucleotides using the S1P/Edg nucleotide sequence as atemplate so that either the full-length cDNA may be generated by knownRACE techniques, or a portion of the coding region may be generated bythese same known RACE techniques to generate and isolate a portion ofthe coding region to use as a probe to screen one of numerous types ofcDNA and/or genomic libraries in order to isolate a full-length versionof the nucleotide sequence encoding S1P/Edg.

It is readily apparent to those skilled in the art that other types oflibraries, as well as libraries constructed from other cell types-orspecies types, may be useful for isolating an S1P/Edg-encoding DNA or anS1P/Edg homologue. Other types of libraries include, but are not limitedto, cDNA libraries derived from other cells.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have S1P/Edgactivity. The selection of cells or cell lines for use in preparing acDNA library to isolate a cDNA encoding S1P/Edg may be done by firstmeasuring cell-associated S1P/Edg activity using any known assayavailable for such a purpose.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Complementary DNA libraries may also be obtained from numerouscommercial sources, including but not limited to Clontech Laboratories,Inc. and Stratagene.

An expression vector containing DNA encoding an S1P/Edg-like protein maybe used for expression of S1P/Edg in a recombinant host cell. Suchrecombinant host cells can be cultured under suitable conditions toproduce S1P/Edg or a biologically equivalent form. Expression vectorsmay include, but are not limited to, cloning vectors, modified cloningvectors, specifically designed plasmids or viruses. Commerciallyavailable mammalian expression vectors may be suitable for recombinantS1P/Edg expression.

Recombinant host cells may be prokaryotic or eukaryotic, including butnot limited to, bacteria such as E. coli, fungal cells such as yeast,mammalian cells including, but not limited to, cell lines of bovine,porcine, monkey and rodent origin; and insect cells including but notlimited to Drosophila and silkworm derived cell lines.

The nucleotide sequences for the various S1P/Edg receptors are known inthe art. See, for example, the following:

S1P₁/Edg1 Human

Hla, T. and T. Maciag 1990 An abundant transcript induced indifferentiating human endothelial cells encodes a polypeptide withstructural similarities to G-protein coupled receptors. J. Biol Chem.265:9308-9313, hereby incorporated by reference in its entirety.

WO91/15583, published on Oct. 17, 1991, hereby incorporated by referencein its entirety.

WO99/46277, published on Sep. 16, 1999, hereby incorporated by referencein its entirety.

S1P₁/Edg1 Mouse

WO0059529, published Oct. 12, 2000, hereby incorporated by reference inits entirety.

U.S. Pat. No. 6,323,333, granted Nov. 27, 2001, hereby incorporated byreference in its entirety.

S1P₁/Edg1 Rat

Lado, D. C., C. S. Browe, A. A. Gaskin, J. M. Borden, and A. J.MacLennan. 1994 Cloning of the rat edg-1 immediate-early gene:expression pattern suggests diverse functions. Gene 149: 331-336, herebyincorporated by reference in its entirety.

U.S. Pat. No. 5,585,476, granted Dec. 17, 1996, hereby incorporated byreference in its entirety.

U.S. Pat. No. 5,856,443, granted Jan. 5, 1999, hereby incorporated byreference in its entirety.

S1P₃/Edg3 Human

An, S., T. Bleu, W. Huang, O. G. Hallmark, S. R. Coughlin, E. J. Goetzl1997 Identification of cDNAs encoding two G protein-coupled receptorsfor lysosphingolipids FEBS Lett. 417:279-282, hereby incorporated byreference in its entirety.

WO 99/60019, published Nov. 25, 1999, hereby incorporated by referencein its entirety.

U.S. Pat. No. 6,130,067, granted Oct. 10, 2000, hereby incorporated byreference in its entirety.

S1P₃/Edg3 Mouse

WO 01/11022, published Feb. 15, 2001, hereby incorporated by referencein its entirety.

S1P₃/Edg3 Rat

WO 01/27137, published Apr. 19, 2001, hereby incorporated by referencein its entirety.

S1P₂/Edg5 Human

An, S., Y. Zheng, T. Bleu 2000 Sphingosine 1-Phosphate-induced cellproliferation, survival, and related signaling events mediated by GProtein-coupled receptors Edg3 and Edg5. J. Biol. Chem 275: 288-296,hereby incorporated by reference in its entirety.

WO 99/35259, published Jul. 15, 1999, hereby incorporated by referencein its entirety.

WO99/54351, published Oct. 28, 1999, hereby incorporated by reference inits entirety.

WO 00/56135, published Sep. 28, 2000, hereby incorporated by referencein its entirety.

S1P₂/Edg5 Mouse

WO 00/60056, published Oct. 12, 2000, hereby incorporated by referencein its entirety.

S1P₂/Edg5 Rat

Okazaki, H., N. Ishizaka, T. Sakurai, K. Kurokawa, K. Goto, M. Kumada,Y. Takuwa 1993 Molecular cloning of a novel putative G protein-coupledreceptor expressed in the cardiovascular system. Biochem. Biophys. Res.Comm. 190:1104-1109, hereby incorporated by reference in its entirety.

MacLennan, A. J., C. S. Browe, A. A. Gaskin, D. C. Lado, G. Shaw 1994Cloning and characterization of a putative G-protein coupled receptorpotentially involved in development. Mol. Cell. Neurosci. 5: 201-209,hereby incorporated by reference in its entirety.

U.S. Pat. No. 5,585,476, granted Dec. 17, 1996, hereby incorporated byreference in its entirety.

U.S. Pat. No. 5,856,443, granted Jan. 5, 1999, hereby incorporated byreference in its entirety.

S1P₄/Edg6 Human

Graler, M. H., G. Bernhardt, M. Lipp 1998 EDG6, a novelG-protein-coupled receptor related to receptors for bioactivelysophospholipids, is specifically expressed in lymphoid tissue.Genomics 53: 164-169, hereby incorporated by reference in its entirety.

WO 98/48016, published Oct. 29, 1998, hereby incorporated by referencein its entirety.

U.S. Pat. No. 5,912,144, granted Jun. 15, 1999, hereby incorporated byreference in its entirety.

WO 98/50549, published Nov. 12, 1998, hereby incorporated by referencein its entirety.

U.S. Pat. No. 6,060,272, granted May 9, 2000, hereby incorporated byreference in its entirety.

WO 99/35106, published Jul. 15, 1999, hereby incorporated by referencein its entirety.

WO 00/15784, published Mar. 23, 2000, hereby incorporated by referencein its entirety.

WO 00/14233, published Mar. 16, 2000, hereby incorporated by referencein its entirety.

S1P₄/Edg6 Mouse

WO 00/15784, published Mar. 23, 2000, hereby incorporated by referencein its entirety.

S1P₅/Edg8 Human

Im, D.-S., J. Clemens, T. L. Macdonald, K. R. Lynch 2001Characterization of the human and mouse sphingosine 1-phosphatereceptor, S1P₅ (Edg-8): Structure-Activity relationship of sphingosine1-phosphate receptors. Biochemistry 40:14053-14060, hereby incorporatedby reference in its entirety.

WO 00/11166, published Mar. 2, 2000, hereby incorporated by reference inits entirety.

WO 00/31258, published Jun. 2, 2000, hereby incorporated by reference inits entirety.

WO 01/04139, published Jan. 18, 2001, hereby incorporated by referencein its entirety.

EP 1 090 925, published Apr. 11, 2001, hereby incorporated by referencein its entirety.

S1P₅/Edg8 Rat

Im, D.-S., C. E. Heise, N. Ancellin, B. F. O'Dowd, G.-J. Shei, R. P.Heavens, M. R. Rigby, T. Hla, S. Mandala, G. McAllister, S. R. George,K. R. Lynch 2000 Characterization of a novel sphingosine 1-phosphatereceptor, Edg-8. J. Biol. Chem. 275: 14281-14286, hereby incorporated byreference in its entirety.

WO 01/05829, published Jan. 25, 2001, hereby incorporated by referencein its entirety.

Measurement of Cardiovascular Effects

The effects of compounds of the present invention on cardiovascularparameters can be evaluated by the following procedure:

Adult male rats (approx. 350 g body weight) were instrumented withfemoral arterial and venous catheters for measurement of arterialpressure and intravenous compound administration, respectively. Animalswere anesthetized with Nembutal (55 mg/kg, ip). Blood pressure and heartrate were recorded on the Gould Po-Ne-Mah data acquisition system. Heartrate was derived from the arterial pulse wave. Following an acclimationperiod, a baseline reading was taken (approximately 20 minutes) and thedata averaged. Compound was administered intravenously (either bolusinjection of approximately 5 seconds or infusion of 15 minutesduration), and data were recorded every 1 minute for 60 minutes postcompound administration. Data are calculated as either the peak changein heart rate or mean arterial pressure or are calculated as the areaunder the curve for changes in heart rate or blood pressure versus time.Data are expressed as mean±SEM. A one-tailed Student's paired t-test isused for statistical comparison to baseline values and consideredsignificant at p<0.05.

The S1P effects on the rat cardiovascular system are described inSugiyama, A., N. N. Aye, Y. Yatomi, Y. Ozaki, K. Hashimoto 2000 Effectsof Sphingosine-1-Phosphate, a naturally occurring biologically activelysophospholipid, on the rat cardiovascular system. Jpn. J. Pharmacol.82: 338-342, hereby incorporated by reference in its entirety.

Measurement of Mouse Acute Toxicity

A single mouse is dosed intravenously (tail vein) with 0.1 ml of testcompound dissolved in a non-toxic vehicle and is observed for signs oftoxicity. Severe signs may include death, seizure, paralysis orunconciousness. Milder signs are also noted and may include ataxia,labored breathing, ruffling or reduced activity relative to normal. Uponnoting signs, the dosing solution is diluted in the same vehicle. Thediluted dose is administered in the same fashion to a second mouse andis likewise observed for signs. The process is repeated until a dose isreached that produces no signs. This is considered the estimatedno-effect level. An additional mouse is dosed at this level to confirmthe absence of signs.

Assessment of Lymphopenia

Compounds are administered as described in Measurement of Mouse AcuteToxicity and lymphopenia is assessed in mice at three hours post dose asfollows. After rendering a mouse unconscious by CO₂ to effect, the chestis opened, 0.5 ml of blood is withdrawn via direct cardiac puncture,blood is immediately stabilized with EDTA and hematology is evaluatedusing a clinical hematology autoanalyzer calibrated for performingmurine differential counts (H2000, CARESIDE, Culver City Calif.).Reduction in lymphocytes by test treatment is established by comparisonof hematological parameters of three mice versus three vehicle treatedmice. The dose used for this evaluation is determined by tolerabilityusing a modification of the dilution method above. For this purpose,no-effect is desirable, mild effects are acceptable and severely toxicdoses are serially diluted to levels that produce only mild effects.

1. A compound represented by Formula II

or a pharmaceutically acceptable salt or hydrate thereof, wherein: n=0or 1; R³ is selected from the group consisting of: hydrogen andC₁₋₄alkyl, optionally substituted with from one up to the maximum numberof substitutable positions with a substituent independently selectedfrom the group consisting of: halo and hydroxy; each R⁴ is independentlyselected from the group consisting of: halo, C₁₋₄alkyl and C₁₋₃alkoxy,said C₁₋₄alkyl and C₁₋₃alkoxy optionally substituted from one up to themaximum number of substitutable positions with halo.
 2. The compoundaccording to claim 1 wherein n is
 0. 3. The compound according to claim1 wherein n is
 1. 4. The compound according to claim 1 wherein R³ ishydrogen.
 5. The compound according to claim 1 selected from thefollowing table:

or a pharmaceutically acceptable salt of any of the foregoing compounds.